PNA synthons

ABSTRACT

A method is disclosed for the preparation of novel PNA synthons compatible with DNA synthetic reagents and instrumentation. Accordingly, the PNA synthons of this invention are particularly suitable for the preparation of PNA-DNA chimeras, among other oligomers. The PNA synthons are designed to have a protecting group strategy which is orthogonal and allows removal of the protecting groups under mild conditions. Generally, an acid labile protected backbone is coupled to a nucleobase side chain moiety to form the PNA synthon. A novel method for synthesizing the acid labile protected backbone also is described. In addition, novel compositions of matter are disclosed.

This application is a division of application Ser. No. 08/910,552, filedon Aug. 11, 1997, now U.S. Pat. No. 6,063,569, which is a continuationof Ser. No. 08/480,228, filed on Jun. 7, 1995, now abandoned, which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of Peptide Nucleic Acid (PNA)synthesis. More particularly, this invention relates to PNA synthonssuitable for the synthesis and deprotection of PNA-DNA chimeras.

2. Description of the Background Art

Peptide Nucleic Acids (PNAs) are synthetic polyamides which arepromising candidates for the sequence-specific regulation of DNAexpression and for the preparation of gene targeted drugs. See EuropeanPatent applications EP 92/01219 and 92/01220 which are hereinincorporated by reference. PNAs are biopolymer hybrids which possess apeptide-like backbone to which the nucleobases of DNA are attached.Specifically, PNAs are synthetic polyamides comprised of repeating unitsof the amino acid, N-(2-aminoethyl)-glycine, to which the nucleobasesadenine, cytosine, guanine, thymine and uracil are attached through amethylene carbonyl group. Other natural and unnatural nucleobases, suchas pseudo isocytosine, 5-methyl cytosine, pseudouracil, isocytosine,hypoxanthine and 2,6-diaminopurine, among many others, also can beincorporated in PNA synthons (see FIG. 8).

PNAs are now routinely synthesized from monomers (PNA synthons)protected according to the t-Boc/benzyl protection strategy, wherein thebackbone amino group of the growing polymer is protected with thet-butyloxycarbonyl (t-Boc) group and the exocyclic amino groups of thenucleobases, if present, are protected with the benzyloxycarbonyl(benzyl) group. PNA synthons protected using the t-Boc/benzyl strategyare now commercially available but are inconvenient to use because,among other reasons, harsh acidic conditions are required to removethese protecting groups.

The t-Boc/benzyl protection strategy requires very strong acids toremove all of the benzyl side chain nucleobase protecting groups.Typically, PNA oligomers are exposed to hydrofluoric acid ortrifluoromethane sulfonic acid for periods of time often exceeding onehour to completely remove the benzyl side chain protecting groups. Thisharsh acid treatment needed for final deprotection will often decompose,among other acid sensitive moieties, nucleic acids and carbohydrateswhich might be attached to the nucleic acid oligomer. Furthermore, theuse of hazardous acids such as hydrofluoric acid or trifluoromethanesulfonic acid is not commercially embraced in view of safety concernsfor the operators and the corrosive effect on automation equipment andlines. The above described harsh conditions are particularly unsuitablefor the synthesis of nucleic acids since the strong acid deprotectionconditions will at least partially decompose nucleic acids.

In addition, the t-Boc/benzyl protection strategy is not orthogonal butdifferential. A differential strategy is defined as a system ofprotecting groups wherein the protecting groups are removed byessentially the same type of reagent or condition, but rely on thedifferent relative rates of reaction to remove one group over the other.For example, in the t-Boc/benzyl protecting strategy, both protectinggroups are acid labile, with benzyl groups requiring a stronger acid forefficient removal. When acid is used to completely remove the more acidlabile t-Boc protecting groups, there is a potential that a percentageof benzyl groups will also be removed contemporaneously. Specifically,the t-Boc protecting group must be removed from the amino group backboneduring each synthetic cycle so the next monomer can be attached to thebackbone at the free amino site thereby allowing the polymeric chain togrow. The deprotection of the t-Boc amino protected backbone isaccomplished using a strong acid such as trifluoroacetic acid. Duringthis deprotection and subsequent construction of the PNA oligomer,removal of the nucleobase side chain protecting groups, i.e., thebenzyls, is undesirable. However, trifluoroacetic acid is potentiallystrong enough to prematurely deprotect a percentage of the side chainbenzyl groups, thereby introducing the possibility of polymer branchingand reducing the overall yield of desired product.

An orthogonal strategy, on the other hand, removes the protecting groupsunder mutually exclusive conditions, e.g., one group is removed withacid while the other group is removed with base. Breipohl et al. havedescribed an orthogonally protected PNA synthon using9-fluorenylmethyloxycarbonyl (Fmoc) as the backbone protecting group anda triphenylmethyl (trityl) group as the side chain nucleobase protectinggroup. Breipohl et al. 1st Australian Peptide Conference, Great BarrierReef, Australia, Oct. 16-21, 1994. This protection methodology, however,is incompatible with standard nucleic acid synthesis methodology.

Christensen et al. have described orthogonal PNA synthons wherein thet-Boc amino backbone protecting group is removed in strong acid thenreprotected with 9-fluorenylmethyloxycarbonyl (Fmoc), a base labileprotecting group. Christensen, L. et al. “Innovation and Perspectives inSolid Phase Synthesis and Complementary Technologies-Biological andBiomedical Applications,” 3rd SPS Oxford Symposia (1994). Although thisprotection strategy eliminates the potential for premature deprotectionof the exocyclic amino group of the side chain nucleobase, extra stepsare involved in preparation of this monomer. Additionally, strong acidssuch as hydrofluoric acid or trifluoromethane sulfonic acid still arerequired to remove the benzyl side chain protecting groups.

Nucleic acids (DNA and RNA) are now routinely synthesized usingautomated machines, numerous synthesis supports and various protectionchemistries. The following U.S. patents cover a broad range of differingsupports and protection chemistries and are herein incorporated byreference. U.S. Pat. Nos. 5,262,530; 4,415,732; 4,458,066; 4,725,677 (RE34,069); and 4,923,901. Automated equipment and reagents arecommercially available from PerSeptive Biosystems, Perkin Elmer (AppliedBiosystems Division) and Pharmacia. Special 5′-amino synthons aredescribed in Smith et al., Nucleic Acids Res. (1985) 13:2399 and inSproat et al., Nucleic Acids Res. (1987) 15:6181. Special 5′-thiosynthons are described in Sproat et al., Nucleic Acids Res. (1987)15:4837. The reagents described in the above references are suitable foruse on standard DNA synthesis instruments.

The preferred commercial method for nucleic acid synthesis utilizes theabove reagents and methods as generally described by Koester et al. inU.S. Pat. No. 4,725,677 (RE 34,069). Consequently, the preferredsynthons are β-cyanoethyl phosphoramidites having acid labile protectionof the backbone 5′ hydroxyl group and base labile acyl-type protectionof the exocyclic amino groups of the nucleobases.

The preferred acid labile backbone protecting group is4,4′-dimethoxytriphenylmethyl (DMT). DMT is typically chosen because itcan be removed farily rapidly (1-3 mintues) during each synthetic cyclewith solutions containing 14% dichloroacetic acid or trichloroaceticacid in dichloromethane. Protecting groups with increased acid labilitycompared to DMT are susceptible to premature deprotection during theacid catalyzed coupling reactions (tetrazole is typically the acidicspecies). Protecting groups with decreased acid lability compared to DMTrequire longer reaction times and/or harsher reaction conditions forcomplete removal. Generally, harsher acidic deprotection conditions areavoided since the purine nucleobases are particularly susceptible todecomposition in acid. Although the aforementioned problems withprotecting groups and synthetic conditions may be minimal during eachsynthetic cycle, the cumulative effect can generate significantimpurities in oligonucleotide synthesis. Accordingly, as the length ofthe oligonucleotide increases, its purity tends to decrease.

Generally, base labile protecting groups are utilized for protection ofthe exocyclic amino groups of the nucleobases so that an orthogonallyprotected nucleic acid synthon results. The base labile protectinggroups typically remain a part of the growing nucleic acid chain, thenare removed simultaneously with the cleavage of synthesized nucleic acidfrom the solid support. A concentrated ammonium hydroxide solution isoften used for the “deprotection and cleavage step.” Koester et al. usedbase labile acyl-type protecting groups which are usually treated for6-24 hours at elevated temperature (about 55° C.) for complete removalof these nucleobase protecting groups. Other protecting groups have beendeveloped which are removed under the same conditions but in less time(from about 15-60 minutes). Examples of these improved protecting groupsinclude phenoxyacetyl, t-butyl phenoxyacetyl and amindine-typeprotecting groups. While these protecting groups have increased baselability, typically only the time necessary for removal is reduced.

Following from the above discussion, PNA synthons suitable for theconstruction of various nucleic acid oligomers should be compatible withcurrent DNA syntheic methodologies so existing techniques and equipmentcan be utilized. Specifically, an appropriately acid labile protectinggroup for backbone protection and an appropriate base labile protectinggroup for nucleobase protection need to be incorporated into the designof a PNA synthon. The ability to syntheisze PNA synthons to meet theabove requirements would allow various combinations of nucleic acids tobe routinely synthesized and, consequently, allow the expansion ofscientific investigations into the utility of these complex molecules.

Another current limitation on the synthesis of PNA synthons is theformation of the side chain nucleobase protecting group. Generally, theexocyclic amino groups of the nucleobases, e.g., cytosine, adenine, andguanine, are protected as carbamates via reaction with activatedcarbonates or chloroformates. This method of carbamate formation suffersfrom the disadvantage that many chloroformates are unstable or that thechloroformates are not appreciably reactive with the mildly nucleophilicexocyclic amino groups of the nucleobases. Other methods of carbamateformation used for nucleobases include the use of imidazolides and alkylimidazolium salts as acylating agents. See Watkins et al, J. Org. Chem.,(1982) 47:4471-77 and Watkins et al., J. Am. Chem. Soc., (1982)104:5702-08. While imidazolides and alkylated imidazolides appear toovercome some of the difficulties associated with carbamate formation,their widespread use with nucleobases has yet to be reported. Recently,the 4methoxy-triphenylmethyl (MMT) group was presented as anotherexocyclic amino protecting group for PNA synthon side chain nucleobases.Breipohl et al. 1st Australian Peptide Conference, Great Barrier Reef,Australia, Oct. 16-21, 1994. The MMT group, however, is not a carbamateprotecting group.

In addition to the above, the synthesis of a selectively protectedguanine PNA synthon has been elusive. The reported guanine PNA synthonsare protected as O-6-benzyl ethers but optionally possess benzylprotection of the exocyclic 2-amino group. See European PatentApplication EP 92/01219 and U.S. patent applications PCT/US92/10921.Given the relative reactivity of the 6-carbonyl group (enol form) andthe more reactive exocyclic 2-amino group, there is no compelling reasonfor protecting the C6 carbonyl group during PNA synthesis, whereasprotection of the more reactive 2-amino group is preferred.

The benzyloxycarbonyl group has been utilized in DNA synthesis for theprotection of the exocyclic amino groups of the nucleobases cytosine,adenine and guanine. See Watkins et al, J. Org. Chem., (1982) 47:4471-77and Watkins et al., J. Am. Chem. Soc., (1982) 104:5702-08. Nonetheless,the guanine synthon was difficult to prepare because the exocyclic2-amino group of guanine was not reactive toward reagents routinely usedto introduce the benzyl group, such as benzyl chloroformate,benzyloxycarbonyl imidazolide and N-alkylated benzyloxycarbonylimidazole. Consequently, a non-conventional multi-step procedure wasdescribed wherein treatment with phenyl chlorothioformate simultaneouslyprotected both the C6 carbonyl group and the exocyclic 2-amino group.Thereafter, the adduct was converted to a carbamate protected guaninecompound whereby the C6 carbonyl protecting group was subsequentlyremoved. Nonetheless, this indirect method is laborious because itrequires the formation of a carbamate protecting group from the initialadduct and the subsequent deprotection of the C6 carbonyl group.

Suitably protected derivatives of 2-amino-6-chloropurine may beconverted to guanine compounds by displacement of the 6-chloro groupwith oxygen nucleophiles. See Robins et al, J. Am. Chem. Soc. (1965)87:4934, Reese et al., Nucl. Acids Res., (1981) 9: 4611 and Hodge etal., J. Org. Chem., (1990) 56:1553. Indeed, suitably protectedderivatives of 2-amino-6-chloropurine are the starting materialscurrently described for preparation of the reported guanine PNAsynthons. See European Patent Application EP 92/01219 and U.S. patentapplication PCT/US92/10921.

The inventors of PNA describe a guanine synthon having no protection ofthe exocyclic 2-amino group but having the 6 carbonyl group protected asa benzyl ether. See European Patent Application EP 92/01219. Thisprotection strategy is surprising because the more reactive 2-aminogroup will likely react (at least marginally) with the activatedcarboxylic acid group of other PNA monomers, thereby causing branchingof the synthesized polymer. Conversely, the enol, which exists when the6-carbonyl group remains unprotected, is not reactive enough to resultin polymer branching and therefore should require no protection. Thisparticular approach is inconsistent with t-Boc/benzyl protectionstrategy they employed for the other PNA synthons.

In a more recent patent application, the guanine PNA synthon has bothbenzyl protection of the exocyclic 2-amino group and a 6 carbonyl groupprotected as a benzyl ether. See U.S. patent application PCT/US92/10921.As previously discussed, there is no compelling rationale for protectingthe 6-carbonyl group of the guanine PNA synthon. However, protection ofthe 6-carbonyl group enables selective ionization of the exocyclic2-amino group of the guanine heterocycle thereby facilitating thereaction of the ionized 2-amino group with conventional benzylprotecting reagents (e.g. benzyloxycarbonyl imidazole). Nonetheless,protection of the exocyclic 2-amino group occurs on a guanine derivativeadditionally protected at the 6-carbonyl group of the nucleobase. Thus,the resulting synthon has both exocyclic 2-amino group and 6-carbonylgroup protection. Hence, there remains no reported convenient high yieldsynthesis of a guanine PNA synthon having selective carbamate protectionof the exocyclic 2-amino group, wherein the 6-carbonyl group remainsunprotected.

Solid phase peptide synthesis methodology is applicable to the synthesisof PNA oligomers, but often requires the use of harsh reactionconditions unsuitable for DNA synthesis, and, consequently, PNA-DNAchimera synthesis too. In the above-mentioned t-Boc/benzyl protectionscheme, the final deprotection of side-chains and release of the PNAmolecule from the solid support is most often carried out by the use ofstrong acids such as anhydrous hydrofluoric acid (HF) (Sakakibara, etal., Bull. Chem. Soc. Jpn., 1965, 38, 4921), boron tris(trifluoroacetate) (Pless, et al., Helv. Chim. Acta, 1973, 46, 1609),and sulfonic acids such as trifluoromethanesulfonic acid andmethanesulfonic acid (Yajima, et al., J. Chem. Soc., Chem. Comm., 1974,107). This conventional strong acid (e.g., anhydrous HF) deprotectionmethod, produces very reactive carbocations that may lead to alkylationand acylation of sensitive residues in the PNA chain. Suchside-reactions are only partly avoided by the presence of scavengerssuch as anisole, phenol, dimethyl sulfide, and mercaptoethanol. Thus,the sulfide-assisted acidolytic S_(N)2 deprotection method (Tam, et al.,J. Am. Chem. Soc., 1983, 105, 6442 and J. Am. Chem. Soc., 1986, 108,5242), the so-called “low,” which removes the precursors of harmfulcarbocations to form inert sulfonium salts, is frequently employed inpeptide and PNA synthesis, either solely or in combination with “high”methods. Less frequently, in special cases, other methods used fordeprotection and/or final cleavage of the PNA-solid support bond are,for example, such methods as base-catalyzed alcoholysis (Barton, et al.,J. Am. Chem. Soc., 1973, 95, 4501), and ammonolysis as well ashydrazinolysis (Bodanszky, et al., Chem., Ind., 1964, 1423),hydrogenolysis (Jones, Tetrahedron Lett., 1977, 2853 and Schlatter, etal., Tetrahedron Lett., 1977, 2861) ), and photolysis (Rich and Gurwara,J. Am. Chem. Soc., 1975, 97, 1575)).

Based on the recognition that most operations are identical in thesynthetic cycles of solid-phase peptide synthesis (as is also the casefor solid-phase PNA), a new matrix, PEPS, was recently introduced (Berg,et al., J. Am. Chem. Soc., 1989, 111, 8024 and International PatentApplication WO 90/02749) to facilitate the preparation of large numbersof peptides This matrix is comprised of a polyethylene (PE) film withpendant long-chain polystyrene (PS) grafts (molecular weight on theorder of 10⁶). The loading capacity of the film is as high as that of abeaded matrix, but PEPS has the additional flexibility to suit multiplesyntheses simultaneously.

Two other methods proposed for the simultaneous synthesis of largenumbers of peptides also apply to the preparation of multiple, differentPNA molecules. The first of these methods (Geysen, et al., Proc. Natl.Acad. Sci. USA, 1984, 81, 3998) utilizes acrylic acid-graftedpolyethylene-rods and 96-microtiter wells to immobilize the growingpeptide chains and to perform the compartmentalized synthesis. Whilehighly effective, the method is only applicable on a microgram scale.The second method (Houghten, Proc. Natl. Acad. Sci. USA, 1984, 82,5131)utilizes a “tea bag” containing traditionally-used polymer beads. Otherrelevant proposals for multiple peptide or PNA synthesis include thesimultaneous use of two different supports with different densities(Tregear, in “Chemistry and Biology of Peptides,” J. Meienhofer, ed.,Ann Arbor Sci., Publ., Ann Arbor, 1972, pp. 175-178), combining ofreaction vessels via a manifold (Gordman, Anal. Biochem., 1984, 136,397), multicolumn solid-phase synthesis (e.g. Krchnak, et al., Int. J.Peptide Protein Res., 1989, 33, 209), and Holm and Meldal, in“Proceedings of the 20th European Peptide Symposium,” G. Jung and E.Bayer, eds., Walter de Gruyter & Co., Berlin, 1989, pp. 208-210), andthe use of cellulose paper (Eichler, et al., Collect. Czech. Chem.Commun., 1989, 54, 1746).

While the conventional cross-linked styrene/divinylbenzene copolymermatrix and the PEPS supports are presently preferred in the context ofsolid-phase PNA synthesis, a nonlimiting list of examples of solidsupports which may be of relevance are: (1) Particles based uponcopolymers of dimethylacrylamide cross-linked withN,N′-bisacryloylethylenediamine, including a known amount ofN-tertbutoxycarbonyl-beta-alanyl N′-acryloylhexamethylenediamine.Several spacer molecules are typically added via the beta alanyl group,followed thereafter by the amino acid residue moietys. Also, the betaalanyl-containing monomer can be replaced with an acryloyl sarcosinemonomer during polymerization to form resin beads. The polymerization isfollowed by reaction of the beads with ethylenediamine to form resinparticles that contain primary amines as the covalently linkedfunctionality. The polyacrylamide-based supports are relatively morehydrophilic than are the polystyrene-based supports and are usually usedwith polar aprotic solvents including dimethylformamide,dimethylacetamide, N-methylpyrrolidone and the like (see Atherton, etal., J. Am. Chem. Soc., 1975, 97, 6584, Bioorg. Chem. 1979, 8, 351), andJ. C. S. Perkin I 538 (1981)); (2) a second group of solid supports isbased on silica-containing particles such as porous glass beads andsilica gel. One example is the reaction product oftrichloro-[3-(4-chloromethyl)phenyl]propylsilane and porous glass beads(see Parr and Grohmann, Angew. Chem. Internal. Ed. 1972, 11, 314) soldunder the trademark “PORASIL E” by Waters Associates, Framingham, Mass.,USA. Similarly, a mono ester of 1,4-dihydroxymethylbenzene and silica(sold under the trademark “BIOPAK” by Waters Associates) has beenreported to be useful (see Bayer and Jung, Tetrahedron Lett., 1970,4503); (3) a third general type of useful solid supports can be termedcomposites in that they contain two major ingredients: a resin andanother material that is also substantially inert to the organicsynthesis reaction conditions employed. A preferred support of this typeis described in U.S. Pat. No. 5,235,028 which is herein incorporated byreference. One exemplary composite (see Scott, et al., J. Chrom. Sci.,1971, 9, 577) utilized glass particles coated with a hydrophobic,cross-linked styrene polymer containing reactive chloromethyl groups,and was supplied by Northgate Laboratories, Inc., of Hamden, Conn., USA.Another exemplary composite contains a core of fluorinated ethylenepolymer onto which has been grafted polystyrene (see Kent andMerrifield, Israel J. Chem. 1978, 17, 243) and van Rietschoten in“Peptides 1974,” Y. Wolman, Ed., Wiley and Sons, New York, 1975, pp.113-116); and (4) contiguous solid supports other than PEPS, such ascotton sheets (Lebl and Eichler, Peptide Res. 1989, 2, 232) andhydroxypropylacrylate-coated polypropylene membranes (Daniels, et al.,Tetrahedron Lett., 1989, 4345), are suited for PNA synthesis as well.

While the solid-phase technique is presently preferred in the context ofPNA synthesis, other methodologies or combinations thereof, for example,in combination with the solid-phase technique, apply as well: (1) theclassical solution-phase methods for peptide synthesis (e.g., Bodanszky,“Principles of Peptide Synthesis,” Springer-Verlag, Berlin-New York1984), either by stepwise assembly or by segment/fragment condensation,are of particular relevance when considering especially large scaleproductions (gram, kilogram, and even tons) of PNA compounds; (2) theso-called “liquid-phase” strategy, which utilizes soluble polymericsupports such as linear polystyrene (Shemyakin, et al., TetrahedronLett., 1965, 2323) and polyethylene glycol (PEG) (Mutter and Bayer,Angew. Chem., Int. Ed. Engl., 1974, 13, 88), is useful; (3) randompolymerization (see, e.g., Odian, “Principles of Polymerization,”McGraw-Hill, New York (1970)) yielding mixtures of many molecularweights (“polydisperse”) peptide or PNA molecules are particularlyrelevant for purposes such as screening for antiviral effects; (4) atechnique based on the use of polymer-supported amino acid active esters(Fridkin, et al., J. Am. Chem. Soc., 1965, 87, 4646), sometimes referredto as “inverse Merrifield synthesis” or “polymeric reagent synthesis,”offers the advantage of isolation and purification of intermediateproducts, and may thus provide a particularly suitable method for thesynthesis of medium-sized, optionally protected, PNA molecules, that cansubsequently be used for fragment condensation into larger PNAmolecules; (5) it is envisaged that PNA molecules may be assembledenzymatically by enzymes such as proteases or derivatives thereof withnovel specificities (obtained, for example, by artificial means such asprotein engineering), and one also can envision the development of “PNAligases” for the condensation of a number of PNA fragments into verylarge PNA molecules; and (6) since antibodies can be gernated tovirtually any molecule of interest, the recently developed catalyticantibodies (abzymes), discovered simultaneously by the groups of Lerner(Tramantano, et al., Science, 1986, 234, 1566) and of Schultz (Pollack,et al., Science, 1986, 234, 1570), also should be considered aspotential candidates for assembling PNA molecules. There has beenconsiderable success in producing abzymes catalyzing acyl-transferreactions (see for example Shokat, et al., Nature, 1989, 338, 269 andreferences therein). Finally, completely artificial enzymes, veryrecently pioneered by Stewart's group (Hahn, et al., Science, 1990, 248,1544), may be developed to suit PNA synthesis. The design of generallyapplicable enzymes, ligases, and catalytic antibodies, capable ofmediating specific coupling reactions, should be more readily achievedfor PNA synthesis than for “normal” peptide synthesis since PNAmolecules will often be comprised of only four different amino acids(one for each of the four native nucleobases) as compared to the twentynaturally occurring (proteinogenic) amino acids constituting peptides.In conclusion, no single strategy may be wholly suitable for thesynthesis of a specific PNA molecule, and therefore, sometimes acombination of methods may work best.

SUMMARY OF THE INVENTION

Accordingly, it is an object of this invention to provide a convenienthigh yield synthetic pathway to novel PNA synthons which are compatiblewith DNA synthetic reagents and instrumentation, and therefore, suitablefor synthesis of PNA-DNA chimeras. Another object of this invention isto provide PNA-DNA chimeras. Still another object of this invention isto provide a novel synthetic route to an acid labile protected aminobackbone used in the construction of the above PNA synthons.

This invention is a method for a convenient high yield synthesis ofnovel PNA suitable for synthesis of PNA-DNA chimeras. While theinvention will be directed towards PNA-DNA chimeras, a PNA-RNA chimeraor other various combinations are equally accesible using the PNAsynthons and methodology of this invention. A PNA-DNA chimera is anoligomer that is composed of at least one PNA moiety and at least oneDNA moiety. PNA synthons suitable for the synthesis of a PNA-DNA chimerapreferably have orthogonal protection compatible with DNA synthesis,i.e., acid labile protection of the reactive group on the backbone andbase labile protection of the side chain nucleobase groups. In addition,the PNA synthons preferably will have protecting groups with similarlability to the protecting groups used in DNA synthesis to avoid the useof harsh chemical conditions that decompose DNA. As part of thisinvention, a novel synthetic process is described for the synthesis ofan increased acid labile protected amino backbone used in theconstruction of the PNA synthons.

Additional features and advantages of the invention will be set forth inthe description which follows, and, in part will be apparent from thedescription, drawings, and claims, or may be learned by practice of thisinvention.

By the method of this invention, a PNA-DNA chimera is synthesized havingthe general formula:

KLQMN

The letters K and N represent chemical bonds, such as covalent bonds,and the letter Q represents a linker or a chemical bond. One of L and Mis a nucleotide moiety having the formula:

The atom or group represented by G is a secondary nitrogen atom, atertiary nitrogen atom having an alkyl substituent, an oxygen atom or asulfur atom. The group represented by B is a protected or unprotected,natural or unnatural nucleobase. The atom or group represented by D is ahydrogen atom, hydroxyl group, a methoxyl group or a hydroxyl groupwhich is protected by a protecting group.

The other of L and M is a PNA moiety of the formula:

The groups represented by G and B are as defined above. The atom orgroup represented by R⁷ is a hydrogen atom or a side chain of aprotected or unprotected naturally occurring a amino acid. Each of j, gand h is the same or different and is independently zero or or aninteger from one to five.

The PNA-DNA chimeras typically are formed from nucleotide moietieshaving the formula:

and PNA moieties having the formula:

where G, B, D, R⁷, j, g and h are as defined above. The atom or grouprepresented by each of Pn and Pa is a hydrogen atom or a protectinggroup, with one of Pn or Pa representing a hydrogen atom. The grouprepresented by R⁶ is a protective group which can be removed afteroligomer synthesis is complete. The entity L¹ is a leaving group orchemical bond. The entity L² is a hydroxyl group, a leaving group or achemical bond.

Preferably, R⁶ is a group having the formula:

Each of V₁-V₃ is the same or different and is independently selectedfrom hydrogen, methyl or ethyl. The group represented by W is anelectron withdrawing group. Preferred electron withdrawing groupsinclude, but are not limited to, cyano, alkyl sulfonyl, aryl sulfonyl,phenyl and substituted phenyl, such as p-nitrophenyl, o-nitrophenyl andp-alkyl sulfonylphenyl.

Preferrably, L¹ is a halogen, CN, SCN or a secondary amino group havingthe formula:

—NR⁸R⁹

where each of R⁸ and R⁹ is the same or different and is independentlyselected from primary, secondary or tertiary alkyl groups having 1-10carbons atoms, or are together selected from cycloalkyl groups having5-7 carbon atoms which can contain one or two nitrogen, oxygen or sulfuratoms as heteroatoms.

The entity L², if a leaving group, can be an activated ester, otherleaving groups such as imidazole, triazole, tetrazole,3-nitro-1,2,4-triazole, thiazole, pyrrole, benzotriazole,benzohydroxytriazole. These cycloalkyl groups also include imdidazolesubstituted in the phenyl moiety, triazole substituted in the phenylmoiety, tetrazole substituted in the phenyl moiety,3-nitro-1,2,4-triazole substituted in the phenyl moiety, thiazolesubstituted in the phenyl moiety, pyrrole substituted in the phenylmoiety, benzotriazole substituted in the phenyl moiety, orbenzohydroxytriazole substituted in the phenyl moiety.

Examples of nucleobase compounds represented by B are shown in FIG. 8.The preferred nucleobases include adenine, cytosine, guanine, thymine,uracil, pseudo isocytosine, 5-methyl cytosine, hypoxanthine,isocytosine, pseudouracil and 2,6-diaminopurine. Generally, nucleobaseshave exocyclic amino groups which are protected during the synthesis ofthe nucleic acid oligomer by removable protecting groups (theseprotected nucleobases are a subset of B and are independentlyrepresented by Ba). The generally accepted orthogonal strategy for DNAsynthesis dictates that the exocyclic amino groups are protected by baselabile protecting groups.

Base labile protecting groups can include base labile carbamateprotecting groups such as an ethoxycarbonyl group having the formula:

The group represented by W is an electron withdrawing group. The atom orgroup represented by each of R²-R⁴ is the same or different and isindependently selected from hydrogen, methyl, ethyl, n-propyl,isopropyl, n-butyl or t-butyl. Preferred electron withdrawing groupsinclude, but are not limited to, cyano, alkyl sulfonyl, aryl sulfonyl,phenyl and substituted phenyl, such as p-nitrophenyl, o-nitrophenyl andp-alkyl sulfonylphenyl.

As an oligomer is synthesized, often with one end of the oligomericchain attached to a solid support, the number of nucleotide or PNAmoieties in the oligomer chain is increased one by one until the desiredsequence is attained. Subsequent nucleotide or PNA moieties having abackbone protecting group are coupled to the previously added moiety onthe growing chain. By the method of this invention, the backboneprotecting group in the orthogonal strategy is an acid labile protectinggroup while the nucleobase is protected by a base labile protectinggroup. Thus, in the case of this invention, the letter K can represent acovalent bond that attaches the nucleic acid oligomer to the nextnucleotide or PNA moiety having a backbone protecting group so as toallow reaction at only one site.

The synthesis of the chimera differs depending on whether Q is acovalent bond or a linker used to connect the PNA and nucleotidemoieties. When a linker is used, the linker is first attached to theterminal PNA or nucleotide moiety followed by a typical coupling cycle.A linker may be used to provide more stable linkages, provide a spacerto optimize the hybridization properties of the chimera, impart aspecial property on the chimera such as to invert polarity or merely forconvenience.

Regardless whether a linker is used, the assembly of the chimeragenerally involves synthesis cycles of a deprotection step followed by acoupling step. When nucleotide moieties are involved, oxidation of thephosphorus atom is an additional required step in the cycle. Inaddition, when coupling nucleotide moieties to a heteroatom, it ispreferable to avoid a nitrogen heteroatom because the resultingnitrogen-phosphorus bond is acid sensitive. Since the linkers usuallyhave a readily accessible functional group and a second protectedreactive group, the same synthetic cycle is often utilized. However,many manipulations of DNA and polypeptide-like moieties are well knownand are suitable for introducing specific reactive functional groupsonto an oligomer without adhering to the synthetic cycle outlined above.

Generally, when Q is a covalent bond, the synthesis cycle will involveproviding a PNA or nucleotide moiety where L¹ or L² is a covalent bond,removing the Pa or Pn protecting group to generate a hydrogen atom onthe heteroatom G, providing a PNA or nucleotide moiety having a Pn or Paprotecting group different from the group just removed, and couplingthis later moiety to the deprotected heteroatom G of the PNA ornucleotide moiety where L¹ or L² is a covalent bond.

On the other hand, when Q is a linker, the synthesis cycle will involveproviding a PNA or nucleotide moiety where L¹ or L² is a covalent bond,removing the Pa or Pn protecting group to generate a hydrogen atom onthe heteroatom G, generating a linker containing a reactive heteroatomby reaction with the heteroatom G, providing a PNA or nucleotide moietyhaving a Pn or Pa protecting group different from the group justremoved, and coupling this later moiety to the heteroatom of the linker.

Typically, at the end of the synthesis, when the desired oligomer isattained, removal of all protecting groups occurs, i.e., removal ofwhichever of Pn or Pa is not hydrogen and removal of the base labileprotecting groups of the nucleobases. If the oligomer was synthesizedusing a solid support, cleavage of the oligomer from the solid supportalso occurs. Because solid phase synthesis is preferred, this inventionalso relates to support bound PNA-DNA chimeras.

PNA-DNA chimeras of this invention may contain a detectable moiety.Examples of detectable moieties include, but are not limited to,enzymes, antigens, radioactive labels, affinity labels, fluorescentlabels, ultraviolet labels, infrared labels and spin labels.

Another aspect of this invention is directed to DNA synthons having theformula:

wherein G, R⁷, j, g, and h are defined above. The group represented byPg is an acid labile protecting group. The group represented by Ba is anatural or unnatural nucleobase having an exocyclic amino groupprotected by a base labile amino protecting group. Examples of Bainclude, but are not limited to, adenine, cytosine, guanine, pseudoisocytosine, 5-methyl cytosine, isocytosine and 2,6-diaminopurine. In apreferred embodiment, G is a secondary nitrogen atom. In other preferredembodiments, Pg and the base labile amino protecting group areindependently carbamate protecting groups.

In another aspect, this invention is directed to PNA synthons having theformula:

wherein Pg and Ba are as defined above.

Preferred embodiments of the compounds occur when Pg is a carbamateprotecting group and specifically the carbamate protecting group havingthe formula:

The atom or group represented by each of A₁-A₁₀ is the same or differentand is independently hydrogen, methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl, hydroxy, methoxy, ethoxy, amide or ester groups. Theester group includes an activated ester. The atom or group representedby R⁴ is hydrogen, methyl or ethyl. The most preferred embodiment occurswhen Pg is 4,4′-dimethylbenzhydroloxycarbonyl.

Preferred embodiments of the above compounds occur when Ba is adenine,cytosine, guanine, pseudo isocytosine, 5-methyl cytosine, isocytosine or2,6-diaminopurine. Preferred embodiments also occur when Ba is protectedby a carbamate base labile protecting group having the formula:

where W and R²-R⁴ are as defined above.

The carbamate base labile protecting group also can be9-fluorenylmethyloxycarbonyl or a 1-cyanoethocycarbonyl group having theformula:

where R²-R⁴ are as defined above. A preferred embodiment occurs when R²is a hydrogen atom and R³ and R⁴ are each methyl groups.

In another embodiment, the carbamate base labile protecting group can bea sulfo-ethoxycarbonyl group having the formula:

where R²-R⁴ are as defined above. The letter n is the integer one ortwo. The group represented by R⁵ is selected from methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, or a substituted or unsubstitutedphenyl group. The substituted or unsubstituted phenyl group has theformula:

where the atom or group represented by each of a-e is the same ordifferent and is independently selected from F, Cl, Br, I, hydrogen,methyl, ethyl, isopropyl, n-propyl, n-butyl, t-butyl, phenyl, methoxy,ethoxy, —NO₂, —CN, —SO₃H, —SCH₃ or —(O)SCH₃. Preferred embodiments occurwhen R⁵ is methyl, n is two and R²-R₄ are each hydrogen; and when R⁵ isan unsubstituted phenyl, n is two and R²∝R⁴ are each hydrogen.

A preferred embodiment of the invention is an adenine PNA synthon havingthe formula:

A preferred embodiment of the invention is a cytosine PNA synthon havingthe formula:

A preferred embodiment of the invention is a guanine PNA synthon havingthe formula:

where n is one or two.

Another preferred embodiment of the invention is a guanine PNA synthonhaving the formula:

where n is one or two.

A preferred embodiment of the invention is a thymine PNA synthon havingthe formula:

A preferred embodiment of the invention is a pseudo isocytosine PNAsynthon having the formula:

In another aspect, the invention is directed to PNA oligomers having thegeneral formula:

KLQMN

where K, Q and N are as previously defined. Each of L and M are PNAmoieties having the formula:

where G, B, R⁷, g, h and j are as previously defined.

Another aspect of the invention is directed to an acid labile protectedbacbone having the general formula:

The group represented by Pg is an acid labile protecting group. The atomrepresented by G is a secondary nitrogen atom, a tertiary nitrogen atomhaving an alkyl substituent, an oxygn atom or a sulfur atom. The entityR⁷ is a hydrogen atom or a side chain of a naturally occurring α aminoacid. The letters g and h are the same or different and areindependently zero or an integer from one to five.

A preferred acid labile protected backbone is an acid labile aminoprotected backbone having the general formula:

The group represented by R⁷ is hydrogen or a side chain of a protectedor unprotected naturally occurring α amino acid. Each of the letters gand h is the same or different and is independently zero or an integerfrom one to five. Preferably, the group represented by Pg is an acidlabile protecting group having the formula:

Each of A₁-A₁₀ is the same or different and is independently hydrogen,methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, hydroxy, methoxy,ethoxy, amide, ester or activated ester groups. The atom or grouprepresented by R⁴ is hydrogen, methyl or ethyl. The preferred embodimentoccurs when R⁴ is a hydrogen atom; A₃ and A₈ are each methyl groups; A₁,A₂, A₄-A₇, A₉ and A₁₀ are each hydrogen atoms; g is one; h is zero; andR⁷ is hydrogen.

Generally, the method of synthesizing the preferred acid labile aminoprotected backbone involves the coupling of an alcohol, e.g., abenzhydrol derivative, to a diamine utilizing a carbonyl equivalentthereby forming a carbamate protecting group on one of the amino groups.After formation of the carbamate protecting group, an unreacted primaryamino group of the diamine is converted to an acetamide derivative whichsubsequently is transformed into a alkyl carboxy group. Otherembodiments of the acid labile protected backbone which include a sulfuratom or oxygen atom as the heteroatom G can be prepared utilizing theabove synthetic methodology.

In another aspect, the invention is directed to the end use of thePNA-DNA chimeras. PNAs exhibit a stronger binding affinity with DNAunder defined conditions which can be exploited for many purposes.Likewise, PNA-DNA chimeras have the potential to exhibit greater bindingaffinity with DNA which increases the potential uses of this uniquesynthetic polymer. Examples of the utility of PNA-DNA chimeras include,but are not limited to, therapeutic or antisense agents, primers inpolymerase reactions and probes for the detection of genetic materialsor sequences.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory and areintended to provide further explanation of the invention as claimed. Theinvention will be understood further from the following drawings, whichare incorporated in and constitute a part of this specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the synthesis of the preferredacid labile protected amino backbone of the amino acidN-(2-aminoethyl)-glycine of this invention.

FIG. 2 is a schematic representation of the synthesis of the preferredcarbamate protected cytosine side chain moiety of this invention.

FIG. 3 is a schematic representation of the synthesis of the preferredcarbamate protected adenine side chain moiety of this invention.

FIG. 4 is a schematic representation of the synthesis of the preferredcarbamate protected guanine side chain moiety of this invention.

FIGS. 5(a) and 5(b) are a schematic representations of the synthesis ofa preferred reagent of this invention for the formation of a carbamateprotecting group.

FIG. 6 is a schematic representation of the coupling of the preferredacid labile protected amino backbone of the amino acidN-(2-aminoethyl)-glycine to the preferred nucleobase side chain moietiesto form the preferred PNA synthons of this invention. The nucleobasesadenine, cytosine, guanine and thymine are shown.

FIG. 7 is a schematic representation of the oxidation of the preferredguanine PNA synthon creating the preferred orthogonally protectedguanine PNA synthon of this invention suitable for PNA-DNA chimerasynthesis.

FIG. 8 is a chart illustrating the structural representations of naturaland unnatural nucleobases useful in this invention.

FIG. 9 is an HPLC trace of the PNA oligomer sequence H₂N-CTTCTCC-CONH₂.

FIG. 10 is an HPLC trace of the PNA oligomer sequenceH₂N-CGCTATACCC-CONH₂.

FIG. 11 is an HPLC trace of the unpurified PNA-DNA chimera sequenceDMBhoc-HN-CACAC-CONH-linker-5′-CCAGT-3′-OH. The underlined portion ofthe sequence represents the PNA sequence and the bold portion of thesequence represents the DNA sequence. DMBhoc is4,4′-dimethylbenzhydroloxycarbonyl.

FIG. 12 is an HPLC trace of the purified PNA-DNA chimera sequenceDMBIboc-HN-CACAC-CONH-linker-5′-CCAGT-3′-OH. The underlined portion ofthe sequence represents the PNA sequence and the bold portion of thesequence represents the DNA sequence. DMBhoc is4,4′-dimethylbenzhydroloxycarbonyl.

FIG. 13 is an HPLC trace of the PNA-DNA chimera sequence (gradient only)DMBhoc-HN-CACAC-CONH-linker-5′-CCAGT-3′-OH. The underlined portion ofthe sequence represents the PNA sequence and the bold portion of thesequence represents the DNA sequence. DMBhoc is4,4′-dimethylbenzhydroloxycarbonyl.

DETAILED DESCRIPTION OF THE INVENTION

The applicant has developed a convenient high yield method for preparingnovel PNA synthons suitable of the synthesis of PNA-DNA chimeras, whichare oligomers composed of at least one PNA moiety and at least one DNAmoiety. Generally, the PNA synthons have orthogonal protection which iscompatible with DNA synthesis, i.e., protecting groups capable ofremoval under mild conditions that will not substantially decompose DNAand having base labile protection of the nucleobase and acid labileprotection of the backbone moiety.

First, the acid labile protected backbone of the PNA synthon issynthesized. Preferably, the acid labile protecting group should havesimilar lability to the most common DNA backbone protecting group,dimethoxytrityl (DMT), the applicants have developed a new syntheticpathway to a preferred acid sensitive backbone.

Second, the nucleobase side chain moiety having base labile protectionof the exocyclic amino groups is synthesized. Either natural orunnatural nucleobases can be incorporated into the PNA synthons.

Third, the acid labile protected backbone is coupled to the nucleobaseside chain moiety which has base labile protection of the exocyclicamino group, if present. The coupling reaction results in PNA synthonssuitable for the synthesis of PNA-DNA chimeras, as well as PNA-RNAchimeras and various other combinations. The PNA synthons of thisinvention also can be used for the synthesis of PNA oligomers under verymild conditions, unlike those currently utilized.

Finally, the PNA synthons of this invention are used in the synthesis ofPNA-DNA chimeras, demonstrating their utility with commerciallyavailable DNA nucleotides. While the description focuses on PNA-DNAchimeras, the PNA synthons and methodologies of this invention areequally applicable to PNA-RNA chimeras and various other combinations.

Acid Labile Protected Backbone Synthesis

In one embodiment, the invention is a method for the preparation of anacid labile protected backbone of a PNA synthon. The preferred backboneis an acid labile amino protected backbone (see FIG. 1). The acid labileprotecting group is capable of removal under mild conditions that willnot substantially decompose DNA moieties. Therefore, when the acidlabile backbone is attached to the nucleobase side chain moiety, theresulting PNA synthon is suitable for PNA-DNA chimera synthesis.

The acid labile protected backbone has the general formula:

The group represented by Pg is an acid labile protecting group. The atomrepresented by G is a secondary nitrogen atom, a tertiary nitrogen atomhaving an alkyl substituent, an oxygen atom or a sulfur atom. The entityR⁷ is a hydrogen atom or a side chain of a protected or unprotectednaturally occurring α amino acid. The letters g and h are the same ordifferent and are independently zero or an integer from one to five.

A preferred acid labile protected backbone is an acid labile aminoprotected backbone having the formula:

The entities represented by Pg, R⁷, g and h are as defined above.Although the synthesis of the preferred acid labile amino protectedbackbone is discussed below, other embodiments of the acid labileprotected backbone including, but not limited to, an oxygen atom orsulfur atom as G are equally accessible by the general syntheticmethodology disclosed in this invention.

Step 1

The first step in the synthesis of the acid labile protected backbone isthe selection of the appropriate alcohol that will be incorporated intothe protecting group. The alcohol may be commercially available or maybe synthetically prepared. The lability of the protecting group willdepend on the stability and ease of formation of the cation of thedehydrated alcohol in an acidic solution. This information is well knownfor many alcohols and is readily available to those skilled in the art.See Sieber et al., Helv. Chemica Acta (1968) 51:614-22.

Examples of alcohols that will provide the required acid lability are,among others, benzhydrol derivatives having the formula:

Each of A₁-A₁₀ is the same or different and is independently hydrogen,methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, hydroxy, methoxy,ethoxy, amide, ester or activated ester groups. The atom or grouprepresented by R⁴ is hydrogen, methyl or ethyl. The atom or grouprepresented by R⁴ is hydrogen, methyl or ethyl. Alkyl groups include,but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl andt-butyl. Alkoxy groups include, but are not limited to, methoxy andethoxy. The preferred embodiment occurs when R⁴ is a hydrogen atom, A₃and A₈ are each methyl groups, and A₁, A₂, A₄-A₇, A₉ and A₁₀ are eachhydrogen atoms.

With reference to FIG. 1, the preferred alcohol is synthesized via aGrignard reaction. First, the magnesium salt of 4-bromotoluene, theGrignard reagent, is prepared in an anhydrous non-nucleophilicether-based solvent. Examples of suitable solvents include, but are notlimited to, diethyl ether, diisopropylether, dioxane andtetrahydrofuran. The preferred solvent is tetrahydrofuran. Afterpreparation of the Grignard reagent, para-tolualdehyde was added to theroom temperature solution of the magnesium salt at a rate sufficient tocreate a reflux. A sufficient time was allowed for the addition of theGrignard reagent to the aldehyde derivative, then the reaction wasquenched with a proton source. Following work up of the reactionmixture, 4,4′- dimethylbenzhydrol is isolated (compound I).

Step 2

In a preferred embodiment, the appropriately selected alcohol isconverted into a corresponding acid labile amino protected diaminehaving the general formula:

The group represented by Pg is an acid labile protecting group. Theletter g is the integer zero or an integer from one to five. Followingthe synthetic methodology described below, Pg necessrily is a carbamateprotecting group.

Generally, the alcohol is reacted with a carbonyl equivalent followed byreaction with a diamine of the formula:

The letter g is zero or an integer from one to five. Examples ofcarbonyl equivalents include, but are not limited to phosgene, phosgenederivatives, carbonyldiimidazole and di-N-succinimidyl carbonate.

With reference to FIG. 1, the preferred carbonyl equivalent iscarbonyldiimadizole (CDI). The preferred diamine occurs when g is one,i.e., is ethylene diamine. Thus, 4,4′-dimethylbenzhydrol is added to asolution of CDI in an anhydrous non-nucleophlic solvent at about 0° C.Examples of anhydrous non-nucleophilic solvents are diethyl ether,diisopropylether, dioxane, tetrahydrofuran, acetonitrile,dichloromethane, chloroform, carbon tetrachloride, benzene and toluene.The preferred solvent is dichloromethane. After the reaction is allowedto occur for a sufficient time, which may be monitored by thin layerchromatography (tlc), the reaction mixture is washed with water, dried,and the dichloromethane evaporated to yield a solid.

The solid is redissolved in an anhydrous non-nucleophilic solvent aspreviously described. Dichloromethane is the preferred solvent. Thedichloromethane solution of the solid is added to the amino backbonemoiety, ethylene diamine, stirring at about 0° C. After the reaction iscomplete, the reaction mixture is washed with water, dried andN-(4,4′-dimethylbenzhydroloxycarbonyl)-ethylenediamine is isolated(compound 11). Thus, the preferred acid labile amino protected diaminehas the formula:

Step 3

In a preferred embodiment, following formation of the acid labile aminoprotected diamine, the remaining primary amino group of the diamine isconverted into an acetamide derivative producing a fully protecteddiamine compound having the general formula:

The entities Pg and g are as defined above. The atom represented by X isan electronegative atom or group. Examples of electronegative atomsinclude, but are not limited to, halogen atoms such as fluorine,chlorine, bromine and iodine.

Generally, the acid labile amino protected diamine is reacted with analkyl trihaloacetate to produce the fully protected diamine compound.Preferably, a non-nucleophlic base is added to neutralize any acidformed by the decomposition of the alkyl trihaloacetate. Examples ofnon-nucleophilic bases include, but are not limited to, triethylamine,diisopropylamine, N-methyl morpholine and N-ethyl morpholine. Thepreferred non-nucleophilic base is triethylamine. Among the variousexamples of alkyl trihaloacetate derivatives, which may include, but arenot limited to, methyl and ethyl esters, ethyltrifluoracetate ispreferred. Accordingly, the preferred atom represented by X is fluorine.

Therefore, the preferred fully protected diamine is prepared by reactionof compound II with ethyl trifluoracetate in dichloromethane at about 0°C. in the presence of triethylamine (see FIG. 1). The product isolatedfrom this reaction isN-[N′-4,4′-dimethylbenzhydroloxycarbonyl-(2′-aminoethyl)]glycine whichhas the formula:

Step 4

The final step in the preparation of the acid labile amino protectedbackbone is the conversion of the acetamide functionality into an alkylcarboxy group. Since the acetamide functionality contains the electronwithdrawing trihalo substituent, the secondary nitrogen proton containedin the acetamide functionality is susceptible to removal by a base toproduce a nucleophilic nitrogen anion. Examples of bases useful for thistransformation include, but are not limited to, lithium hydride, sodiumhydride and potassium hydride. The preferred base is sodium hydride.

Following removal of the secondary nitrogen proton, an alkylhaloalkylate is added and the reaction mixture allowed to stir until thereaction is complete. Examples of alkyl haloalkylates, among others, aremethyl chloroacetate, ethyl chloroacetate, methyl bromoacetate, ethylbromoacetate, methyl iodoacetate, ethyl iodoacetate, methyl3-chloropropionate, ethyl 3-chloropropionate, methyl 3-bromopropionate,ethyl 3-bromopropionate, methyl 3-iodopropionate and ethyl3-iodopropionate. The preferred compound is ethyl bromoacetate.Additionally, the alkyl haloalkylate may be substituted at the halocontaining carbon atom with a side chain of a protected or unprotectednaturally occurring α amino acid.

A crude intermediate product is isolated, then treated with a hydroxideion source. Examples of hydroxide ion sources include, but are notlimited to, lithium hydroxide, sodium hydroxide and potassium hydroxide.The preferred hydroxide ion source is lithium hydroxide. Subsequent towork-up, an acid labile amino protected backbone is isolated having thegeneral formula:

The entities Pg and g are as defined above. The letter h is zero or aninteger from one to five and R⁷ is hydrogen or a side chain of aprotected or unprotected naturally occurring a amino acids. Withreference to FIG. 1, the preferred acid labile amino protected backbonehas the formula:

Nucleobase Side Chain Moiety Synthesis

In another embodiment, the invention is directed to the synthesis ofnucleobase side chain moieties for coupling to the acid labile aminoprotected backbone (see FIGS. 2-5).

Since the PNA synthons of this invention are orthogonally protected, theexocyclic amino groups of the nucleobase side chain moieties necessarilyare not protected with acid labile protecting groups. Examples ofpossible protecting groups include, but are not limited to, those thatare cleaved by basic conditions, photolytic conditions or hydrogenolysisconditions. The preferred nucleobase protecting groups are base labile.Generally, the novel nucleobase side chain moieties have the generalformula:

The letter j is zero or an integer from one to five. The entity Ba is anatural or unnatural nucleobase having an exocyclic amino groupprotected by a base labile protecting group (as described above, Ba is asubset of B). Examples of natural nucleobases having an exocyclic aminogroup are adenine, cytosine and guanine. Examples of unnaturalnucleobases having an exocyclic amino group include, but are not limitedto, pseudo isocytosine, 5-methyl cytosine, isocytosine and2,6-diaminopurine.

Examples of base labile amino protecting groups include, but are notlimited to, 9-fluorenylmethyloxycarbonyl (Fmoc) and ethoxycarbonylgroups having the general formula:

The group represented by W is an electron withdrawing group, The atom orgroup represented by each of R²-R⁴ is the same or different and isindependently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl ort-butyl.

The novel nucleobase side chain moieties of this invention can besynthesized via a novel isocyanate method, as disclosed in a companionpatent application entitled “Improved Synthons for the Synthesis andDeprotection of Peptide Nucleic Acids Under Mild Conditions” (U.S.application Ser. No. 08/487,666, filed Jun. 7, 1995) which is hereinspecifically incorporated by reference in its entirety. Alternatively,the nucleobase side chain moieties can be synthesized by conventionalmethods which involve protection of the exocyclic amino groups of thenucleobase with imidazolides or alkyl imidazolium salts.

Step 1

In both of the above synthetic schemes, a nucleobase is firsttransformed into a partially protected nucleobase compound having analkyl formate substituent, an alkyl acetate substituent or an alkylester substituent such as an alkyl propionate, an alkyl butanoate, analkyl pentanoate or an alkyl hexanoate. Thus, the partially protectednucleobase compound has the formula:

The letter j is zero or an integer from one to five. The entity B is thenucleobase having an exocyclic amino group. The group represented by R¹⁰is methyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)-ethyl,2-(phenylthio)-ethyl, propyl, isopropyl, n-butyl, t-butyl, allyl,1-isopropyl allyl, cinnamyl, 4-nitrocinnamyl, or a substituted orunsubstituted benzyl.

Generally, sequential treatment of the nucleobase with a base such aspotassium carbonate or potassium t-butoxide and an alkyl haloalkylateproduces the partially protected nucleobase compound. An alkyl or benzylbromoacetate produces the preferred partially protected nucleobasecompounds.

With reference to FIG. 2, the preferred partially protected cytosinecompound has the formula:

With reference to FIG. 3, the preferred partially protected adeninecompound has the formula:

With reference to FIG. 4, the preferred partially protected guanineprecursor compound has the formula:

Step 2

The partially protected nucleobase compound is transformed into a fullyprotected nucleobase compound by protecting the exocyclic amino group ofthe nucleobase with a base labile protecting group. The fully protectedbase labile protected compound has the general formula:

The letter j is as previously defined. The groups represented by Ba andR¹⁰ are as previously defined. The base labile protecting group can beformed by either the isocyanate method as referenced above, e.g., aswith the partially protected guanine compound, or by conventionalmethods, e.g., as with the partially protected adenine and cytosinecompounds.

Examples of base labile amino protecting groups include, but are notlimited to, 9-fluorenylmethyloxycarbonyl (Fmoc) and ethoxycarbonylgroups having the general formula:

The group represented by W is an electron withdrawing group. The atom orgroup represented by each of R²-R⁴ is the same or different and isindependently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl ort-butyl.

Examples of electron withdrawing groups are, among others, cyano, alkylsulfonyl, aryl sulfonyl, phenyl and substituted phenyl such asp-nitrophenyl, o-nitrophenyl and p-alkyl sulfonyl phenyl. With CN as theelectron withdrawing group, the base labile protecting group has theformula:

The atom or group represented by each of R²-R⁴ is the same or differentand is independently hydrogen, methyl, ethyl, n-propyl, isopropyl,n-butyl or t-butyl.

The preferred method of preparing the 1-cyanoethoxycarbonyl group is byreaction of the corresponding 1-cyanoethanol derivative withcarbonyldimidazole to form the imidazolide salt. The preferred1-cyanoethanol derivative is 2-hydroxy-2-methyl-butyronitrile. Thus, inreference to FIG. 5a, reaction of 2-hydroxy-2-methyl-butyronitrile withcarbonyldiimidazole produces compound XIV,2,2-dimethyl-1-cyanoethoxycarbonyl-imidazole.

To enhance the acylating efficiency of this preferred reagent,2,2-dimethyl-1-cyanoethoxycarbonyl-imidazole is reacted with methyltrifluoromethanesulfonate to produce compound XV (see FIG. 5b).Preferably, the reagent is generated in situ immediately before reactionwith the partially protected nucleobase compound.

Thus, with reference to FIG. 2, the preferred fully protected cytosinecompound has the formula:

With reference to FIG. 3, the preferred fully protected adenine compoundhas the formula:

With a sulfoxide or sulfone as the electron withdrawing group, the fullyprotected nucleobase compound is typically formed via the isocyanatemethod and has the formula:

The letter n is one or two. The atom or group represented by each ofR²-R⁴ is the same or different and is independently hydrogen, methyl,ethyl, n-propyl, isopropyl, n-butyl or t-butyl. The group represented byR⁵ is methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl or asubstituted or unsubstituted phenyl having the formula:

The atom or group represented by each of a-e is the same or differentand is independently F, Cl, Br, I, hydrogen, methyl, ethyl, isopropyl,n-propyl, n-butyl, t-butyl, phenyl, methoxy, ethoxy, —NO₂, —CN, —SO₃H,—SCH₃ or —(O)SCH₃.

Of special note, in the case of guanine, the exocyclic amino group of2-amino-6-chloropurine is first protected as a thioether group, i.e., anunoxidized sulfur (see FIG. 4, compound XII). Although not a base labileprotecting group, the thioether group avoids premature base eliminationof the sulfur containing protecting group since preparation of thecarbamate protected guanine side chain moiety (compound XIII) requirestreatment with strong base. Only after coupling to the acid labile aminoprotected backbone is the sulfur atom oxidized to form the base labileprotecting group.

Thus, with reference to FIG. 4, the preferred fully protected guanineprecursor compound has the formula:

Step 3

Generally, the acetate ester group of the fully protected nucleobasecompound is hydrolyzed with acid to produce the nucleobase side chainmoiety. In the case of the fully protected guanine compound, hydrolysisis contemporaneous with formation of the 6-carbonyl group as describedin “Improved Synthons for the Synthesis and Deprotection of PeptideNucleic Acids Under Mild Conditions” (U.S. application Ser. No.08/487,666, filed Jun. 7, 1995) (see FIG. 4 of this application).

With reference to FIG. 2, the preferred cytosine side chain moiety hasthe formula:

With reference to FIG. 3, the preferred adenine side chain moiety hasthe formula:

With reference to FIG. 4, the preferred guanine side chain moiety hasthe formula:

Other nucleobase side chain moieties include a thymine side chain moietyand an uracil side chain moiety. Both of these nucleobases lackexocyclic amino groups, therefore, do not require the additionalprotection steps. However, the thymine side chain moiety and the uracilside chain moiety can be coupled to the acid labile amino protectedbackbone to form novel PNA synthons.

PNA Synthon Synthesis (“The Coupling Step”)

In another embodiment, the invention is a method for the preparation ofPNA synthons suitable for PNA-DNA chimera synthesis (see FIG. 6). Thepreparation of a guanine PNA synthon having a thioether group protectingthe exocyclic amino group of guanine involves the oxidation of thethioether group to a sulfoxide or sulfone derivative (see FIG. 7). Theresultant sulfoxide or sulfone transforms the thioether protecting groupinto a base labile protecting group, thus creating an orthogonallyprotected guanine PNA synthon.

Coupling of an acid labile protected backbone to the nucleobase sidechain moiety produces a PNA synthon having the formula:

The entities Pg, Ba, R⁷, g, h and j are as previously defined. The grouprepresented by G is a secondary nitrogen atom, a tertiary nitrogen atomhaving an alkyl substituent, an oxygen atom or a sulfur atom.

Because the PNA synthon is orthogonally protected, i.e., typically oneacid labile protecting group and one base labile protecting group, themethod of coupling the two moieties together preferably minimizes theconditions that will remove either of the two protecting groups.Previously described coupling methods often used the carboxylic ester ofa protected backbone, then hydrolyzed the ester with a hydroxide ionsource. The basic hydrolysis conditions are unsuitable for the PNAsynthons of this invention as the base labile protecting group of thenucleobases would be at least partially removed. Therefore, thepreferred method of coupling the nucleobase side chain moiety to theacid labile protected backbone involves “transient” protection of thecarboxylic acid functionality of the protected backbone.

Generally, the coupling method involves addition of a silyl carboxylicacid protected acid labile protected backbone to a solution of apreformed mixed anhydride of the nucleobase side chain moiety. Followingcoupling of the backbone and the side chain moiety, the silyl protectinggroup is removed to produce the PNA synthon.

More specifically, the mixed anhydride of the nucleobase side chainmoiety is formed by treatment of the nucleobase side chain moiety with anon-nucleophilic base at below ambient temperature, followed by reactionwith a alkyl acid chloride. Non-nucleophilic bases useful in this stepof the reaction sequence include, but are not limited to, triethylamine,diisopropylethylamine, N-methyl morpholine and N-ethyl morpholine. Thepreferred non-nucleophilic base is N-methyl morpholine. Typically thereaction is stirred at below ambient temperature, preferably about 0°C., for a sufficient time to allow formation of the mixed anhydride,which may be monitored by tlc.

Following formation of the mixed anhydride, a solution of the acidlabile protected backbone in the presence of a non-nucleophilic base anda sterically hindered silyl chloride is added to the cooled mixedanhydride solution. Typically, the solution of the acid labile aminoprotected backbone also is below ambient temperature, preferrable about0° C. Examples of non-nucleophilic bases are those previously described.A preferred sterically hindered silyl chloride is triisopropylsilylchloride, however, other silyl chlorides can be used. After the reactionis stirred for a sufficient time to allow coupling, the reaction isquenched, dried and then subjected to treatment with a silyl removinggroup in the presence of a non-nucleophilic base. Again, examples ofnon-nucleophilic bases are those previously described. Examples of silylremoving groups include, but are not limited to, hydrogen fluoride,tetra-n-butyl ammonium fluoride and triethylamine trihydrofluoride. Thepreferred silyl removing group is triethylamine trihydrofluoride becausethe pH of the solution can be adjusted, thereby minimizing prematureremoval of other protecting groups. Subsequent to removal of the silylprotecting group and work-up of the reaction, the desired PNA synthon isisolated.

Thus, in one embodiment, a preferred PNA synthon has the formula:

The groups Pg and Ba are as previously defined.

In another embodiment, a preferred PNA synthon has the formula:

The group represented by B is as previously defined. The grouprepresented by Pg is an acid labile protecting group of the formula:

Each of A₁-A₁₀ is the same or different and is independently hydrogen,methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, hydroxy, methoxy,ethoxy, amide or ester groups. The atom or group represented by R⁴ ishydrogen, methyl or ethyl.

In another embodiment, the preferred cytosine PNA synthon has theformula:

In another embodiment, the preferred adenine PNA synthon has theformula:

In another embodiment, a preferred guanine PNA synthon has the formula:

The group represented by R⁵ is methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl or a substituted or unsubstituted phenyl group havingthe formula:

where the atom or group represented by each of a-e is the same ordifferent and is independently F, Cl, Br, I, hydrogen, methyl, ethyl,isopropyl, n-propyl, n-butyl, t-butyl, phenyl, methoxy, ethoxy, —NO₂,—CN, —SO₃H, —SCH₃ or —(O)SCH₃. It should be noted that the guanine PNAsynthon can be oxidized to generate the base labile protecting groupbefore or after incorporation into an oligomer.

In another embodiment, the preferred thymine PNA synthon has theformula:

In another embodiment, the preferred pseudo isocytosine PNA synthon hasthe formula:

To complete the synthesis of a preferred guanine PNA synthon having athioether containing protecting group, oxidation of the thioether groupremains. Methods of oxidizing a sulfur atom to a sulfoxide or to a fullyoxidized sulfone are known to those skilled in the art. Tesser et al.,Int. J. Peptide Protein Res. (1975) 7:295-305. For example, a preferredmethod involves the sequential treatment of the guanine synthon(compound XVIII) in an aqueous solvent with a non-nucleophilic base,sodium tungstate, and hydrogen peroxide (see FIG. 7). Typically thereaction is monitored for completeness by tlc or high performance liquidchromatography (HPLC). After the reaction is complete, work-up andisolation of the product produces the preferred guanine PNA synthonhaving the formula:

Thus, in one embodiment, a preferred guanine PNA synthon has theformula:

The letter n is one or two.

In another embodiment, a preferred guanine PNA synthon has the formula:

The letter n is one or two.

PNA-DNA Chimera or PNA Oligomer Synthesis

In another embodiment, the invention is a method for the preparation ofa PNA-DNA chimera or a PNA oligomer. Typically, for the efficientsynthesis of these nucleic acid polymers, mild reaction conditions arerequired and an orthogonal protecting group strategy is used. Therefore,the PNA synthons of this invention are particularly advantageous becausetheir protecting groups are removed under mild conditions and areorthogonal when two protecting groups are required. Moreover, theprotection methodology is compatible with commercial DNA synthesistechnology.

Generally, the PNA-DNA chimera (or a PNA-RNA chimera and variouscombinations thereof) will have the formula:

KLQMN

The letters K and N represent chemical bonds. The letter Q is a chemicalbond or a linker. One of L and M is a nucleotide moiety having theformula:

The atom represented by G is a secondary nitrogen atom, a tertiarynitrogen atom having an alkyl substituent, an oxygen atom or a sulfuratom. The entity B is a protected or unprotected, natural or unnaturalnucleobase. The atom or group represented by D is a hydrogen atom, ahydroxyl group, a methoxyl group or a hydroxyl group which is protectedby a protecting group.

The other of L and M is a PNA moiety having the formula:

The entities G and B are as defined above. The entity R⁷ is a hydrogenatom or a side chain of a protected or unprotected naturally occurring αamino acid. Each of j, g and h is the same or different and isindependently zero or an integer from one to five.

The chemical bond defined by K and N can be a covalent bond or an ionicbond. The chemical bond can attach a PNA moiety or a nucloetide moietyto another PNA moiety or nucleotide moiety, either as a single speciesor as part of a polymeric chain. The chemical bonds also can attachlinkers, linkers coupled to a solid support, or other chemical entities.Examples of chemical entities include, but are not limited to,protecting groups, hydrogen atoms, alkyl groups and aryl groups as onlya few examples. Examples of solid supports include, but are not limitedto, controlled pore glass, membranes, beaded polystyrene, silica gel,silica, papaer fitters and fritted glass. One of skill in the art willbe able to further identify and expand the possibilities of otherchemical entities, supports and bonds that would be appropriate anduseful in this invention.

Where Q represents a chemical bond, a covalent bond is intended. As withK and N, the chemical bond can attach a PNA moiety or a nucloetidemoiety to another PNA moiety or nucleotide moiety, either as a singlespecies or as part of a polymeric chain. The entity Q also can representa linker which connects the PNA moiety and the nucleotide moiety.

Linkers are well known in the field of DNA and peptide synthesis. For adetailed discussion, see U.S. Pat. No. 5,410,068 which in hereinincorporated by reference and Gati, M. J., Oligonucleotide Synthesis, APractical Approach, IRL Press Inc., Oxford, England. As used herein, alinker is a composition which can be used for attachment of a moiety toa biopolymer. The moiety may be a support, a biomolecule such as anamino acid or nucleotide or a label such as a fluorescent dye. As usedherein, the linker Q is used to modify the heteroatom G of the PNA ornucleotide moiety.

One type of linker comprises a phosphoramidite linker which typically isused to label the terminus of a synthetic oligonucleotide. The linkercomprises a reactive phosphoramidite group a spacer and at least oneprotected heteroatom. Examples of such preformed DNA linkers includethose described in the following references. Nelson et al., Nucleic AcidResearch, (1989) 17, 7170; Misiura et al, Nucleic Acids Research (1990)18, 4345; Connelly, B., Nucleic Acids Research (1987) 15, 3131; Coull etal., Tetrahedron Letters (1986) 27, 3991. The spacer is generally analkyl spacer having 1-12 carbon atoms. The protecting group of theheteroatom is chosen to be compatible with DNA synthesis. Linkers orthis type are generally commercially available from suppliers of DNAsynthesis reagents.

The 5′-terminus of an oligonucleotide likewise can be modified by knownmethods. This method is likewise suitable for modifying the terminus ofa PNA. See Wachter et al., Nucleic Acids Res. (1986)14:7985. Generallythe heteroatom of the terminus is reacted sequentially withcarbonyldiimidazole and then with an amine containing compound ofchoice. The amine containing compound for the purposes of chimerasynthesis will preferably include alkyl diamines, amino alkane thiolsand amino alkyl alcohols having 1-12 carbons. Suitable reagents areavailable from Aldrich Chemical. Using the above referenced reagents andmethods, one can modify the terminus of a nucleotide, or otherheteroatom, to thereby introduce a functional linkage to the originalheteroatom, an alkyl spacer and terminal heteroatom of choice which canbe used to couple the next synthon of the PNA oligomer or the PNA ornucleotide moiety of the PNA/DNA chimera.

Finally, a new linker has been described for reversibly labelingbiopolymers. See U.S. Pat. No. 5,410,068 which is herein incorporated byreference. Though primarily directed at DNA applications, the teachingsof the above patent can be utilized to link PNA and DNA monomersubunits. This linker has an active ester and stereoselective alkylhalide reactive center. Persons of ordinary skill will recognize thatsuch a versatile reagent can be utilized in numerous ways to link themonomer subunits together.

In one embodiment, the hydroxyl or thiol terminus of the terminalsubunit will react preferentially with the stereoselective alkyl halidecenter. The active ester is then preferentially reacted with an aminecontaining compound as described above to generate a new terminalheteroatom which can be used to couple the next synthon of the PNAoligomer, PNA moiety or DNA nucleotide of the PNA/DNA chimera.Alternatively, a terminal amino group will preferentially react with theactive ester. Then the stereoselective alkyl halide reactive center canbe reacted preferably with an alkyl diamine, alkyl diol, or alkyldithiol to thereby introduce a terminal heteroatom suitable for couplingthe next synthon of the PNA oligomer or the PNA moiety or nucleotidemoiety of the PNA/DNA chimera.

Use of the above described linker also is advantageous because inaddition to the above described reagents (e.g., alkyl diamine, alkyldiol, or alkyl dithiol) the compound may also be a PNA moiety ornucleotide moiety wherein Pn or Pa in a hydrogen atom. In thisembodiment one is able to invert to polarity of the two subunits. Thismay be useful since it is known that PNA can bind to DNA in bothparallel and antiparallel motifs. See Egholm et al., Nature (1993) 365,566-568.

Other linkers have been utilized in peptide synthesis. Generally theselinkers are described for use in manipulating the appropriate heteroatomfunctional groups on biomolecules, supports and labeling reagents, butalso have utility in other areas. Discussion of these linkers, as theyrelate to peptide synthesis, can be found in the following U.S. patentswhich are herein incorporated by reference. U.S. Pat. Nos. 5,117,009;5,306,562; 5,196,566; and 5,187,625. One of ordinary skill inoligonucleotide synthesis, peptide synthesis and PNA synthesis willrecognize these linkers as suitable for the linking of subunits of PNAsto each other or alternatively for the linking of PNA subunits to DNAand conversely the linking of DNA subunits to PNA.

Protected or unprotected, natural or unnatural nucleobases typically arethose previously described or illustrated in FIG. 8. Generally, thenucleobases have an exocyclic amino group that is protected during theconstruction of the PNA-DNA chimera, then is removed after the preferredsequence is attained. On the other hand, the natural nucleobases thymineand uracil lack an exocyclic amino group, thus, usually are notprotected. Protection of the exocyclic amino group of the nucleobase, ifnecessary, eliminates a potential reactive site during oligomersynthesis, and prevents undesired branching of the growing polymericchain. Therefore, as in any efficient synthetic scheme, PNA-DNA chimerascan be assembled in a controlled fashion with only one likely product toresult from each synthetic step.

Typically, the nucleobase protecting groups are carbamate protectinggroups. Since the backbone of the PNA synthons and of the nucleotidesare acid labile, an orthogonal strategy dictates that the nucleobaseprotecting groups necessarily be removable by different means. Asdescribed above in the synthesis of the nucleobase side chain moiety,the preferred carbamate protecting groups for the nucleobases are baselabile.

Often the PNA-DNA chimera will have detectable moieties. The detectablemoieties may be an intrinsic part of the chimera or may be attached tothe chimera in a variety of ways, often through the use of a linker aspreviously described. Examples of detectable moieties include, but arenot limited to, enzymes, antigens, radioactive labels, affinity labels,fluorescent labels, ultraviolet labels, infrared labels and spin labels.The detectable moiety can function, among other purposes, to monitor thesynthetic assembly of the chimera or to monitor the binding of thechimera to another entity such as a DNA or RNA.

The use of labeled biomolecules is well known and is discussed in theabove referenced literature related to linkers. Labeled DNA oligomersare particularly useful for DNA sequencing and other detection analysesof nucleic acids wherein the sequence specific interactions, i.e.,hybridization, of DNA occurs. See U.S. Pat. No. 5,149,625 whichdiscusses labeled DNA and its utility in DNA sequencing applications andis herein incorporated by reference. Labels generally increase thesensitivity of the assay if attached to the biopolymer is a suitablemanner. It is known that antisense DNA and RNA can modulate theexpressions of genes because of these sequence specific interactions.Because PNA exhibits stronger sequence specific interaction with nucleicacids under certain conditions, PNA-DNA chimera are interestingcandidates for further investigation as alternatives in DNA sequencing,detection assays and as therapeutic agents.

Among other methods, PNA-DNA chimeras can be constructed utilizingtechniques for the synthesis of DNA sequences. DNA synthesis methods andinstrumentation are well known in the industry and are widely available.Therefore, the specific design of the PNA synthons of this inventionwill allow exploitation of commercially available DNA precursors andinstrumentation for the straightforward assembly of PNA-DNA chimeras.

Generally, nucleotides commonly used in DNA synthesis and applicable forPNA-DNA chimera synthesis have the formula:

The entities G and B are as previously described. The entity D is ahydrogen atom, a hydroxyl group, a methoxyl group or a hydroxyl groupwhich is protected by a protecting group. The entity Pn is a hydrogenatom or a protecting group. The group R⁶ is a protecting group that canbe removed. The phosphorus linkage is typically oxidized after eachcoupling step to stabilize the linkage. The entity L¹ is a leaving groupor a chemical bond. One example of a nucleotide moiety having the aboveformula, among others, is a phosphoramidite.

Besides nucleotides having the above structure, other nucleotideanalogues that are compatible with a DNA synthesizer can be used to formPNA-DNA chimeras. For examples of DNA synthons, see U.S. Pat. Nos.4,458,066 and 4,725,677 (RE 34,069), and Smith et al., Nucleic AcidsRes. (1985) 13:2399, Sproat et al., Nucleic Acids Res. (1987) 15:6181and Sproat et al., Nucleic Acids Res. (1987) 15:4837.

The selection of D determine whether the nucleotide moiety is a DNA oran RNA moiety. If D is a hydrogen atom, the nucleotide is a DNA moiety.If D is a hydroxyl group or a protected derivative thereof, thenucleotide is an RNA moiety. A methoxyl group is a common substituentemployed in oligomer synthesis. A number of other protecting groupsincluding, but not limited to, silyl protecting groups, are used toprotect the hydroxyl group during oligomer synthesis.

Whether Pn is a hydrogen atom or a protecting group is determined by therole of the nucleotide moiety in that particular synthetic step. As theoligomeric chain is assembled one moiety (or monomer) at a time, eachnew moiety added to the growing chain will have the atom represented byG protected, thereby rendering G unavailable as a reactive site.Therefore, if the nucleotide moiety is the next monomer to be added tothe oligomer, Pn is a protecting group. (Pn will also be a protectinggroup if the nucleotide monomer is the first moiety to be attached toanother monomer or to a solid support resin.) Conversely, if thenucleotide moiety is already part of an oligomeric chain, i.e., is themoiety that will act as the nucleophilic species in the couplingreaction, Pn will be a hydrogen atom.

Suitable R⁶ substituents are described in U.S. Pat. Nos. 4,458,066 and4,725,677, which are herein incorporated by reference. The group R⁶ istypically, among other possibilities, a group of the formula:

The group represented by W is an electron withdrawing group. The atomsor groups represented by each of V₁-V₃ are the same or different and areindependently hydrogen, methyl and ethyl. Preferred electron withdrawinggroups include, but are not limited to, cyano, alkyl sulfonyl, arylsulfonyl, phenyl and substituted phenyl, such as p-nitrophenyl,o-nitrophenyl and p-alkyl sulfonylphenyl.

The group represented by L¹ can be a chemical bond if, for example, thenucleotide moiety is already incorporated into a polymeric chain orattached to a solid support through a linker. The group represented byL¹ also can be a leaving group. Reaction of the preceeding PNA ornucleotide moiety with the nucleotide moiety with Pn as a protectinggroup will displace the leaving group, thereby coupling the two moietiestogether. Suitable leaving groups include active esters as described inU.S. Pat. Nos. 5,233,044 and 5,410,068 which are herein incorporated byreference. Other examples of leaving groups include, but are not limitedto, halogens and secondary amino groups.

The secondary amino groups can have the formula:

—NR⁸R⁹

The groups represented by R⁸ and R⁹ are the same or different and areindependently primary, secondary or tertiary alkyl groups having 1-10carbons atoms, or are together selected from the group consisting ofcycloalkyl groups having 5-7 carbon atoms which can contain one or twonitrogen, oxygen or sulfur atoms as heteroatoms.

PNA moieties used in the construction of PNA-DNA chimeras have theformula:

The entities G, B, R⁷, j, g and h are as previously described. Theentity Pa is a hydrogen atom or a protecting group. The entity L² is ahydroxyl group, a leaving group or a chemical bond.

Typically, as with the nucleotide moiety, the entity Pa is a hydrogenatom if the PNA moiety will act as the nucleophilic species in thecoupling reaction. On the other hand, if the PNA moiety is the monomerto be coupled to a nucleophilic species, then Pa will be a protectinggroup, rendering G unsuitable as a reactive site.

Similarly, the identity of L² also is dependent upon the role of the PNAmoiety in the synthetic sequence. If the PNA moiety is attached to anoligomer or a linker attached to a solid support, L² will be a chemicalbond. However, if the PNA moiety is a monomer to be attached to a linkeror a PNA or nucleotide moiety, L² will be a hydroxyl group or a leavinggroup. As described above for the nucleotide moiety, nucleophilic attackwill displace the leaving group or activated hydroxyl group,facilitating coupling of the PNA moiety to the nucleophilic species.Typically, the coupling step involves the generating the activatedsynthon, i.e., converting the hydroxyl group to a leaving group. Theactivation is usually carried out in situ. Suitable activatingchemistries are well known in the art of peptide chemistry.

The entity L² also may preferably be an active ester such as thosedescribed in U.S. Pat. Nos. 5,410,068 and 5,233,044. Preferrable L² isalso 1-hydroxy-7-azabenzotriazole (HOAT).

Other examples of appropriate L² groups will be readily known to thoseskilled in the art and preferrably include, among others, imidazole,triazole, tetrazole, 3-nitro-1,2,4-triazole, thiazole, pyrrole,benzotriazole, and benzohydroxytriazole. These cycloalkyl groups alsoinclude imdidazole substituted in the phenyl moiety, triazolesubstituted in the phenyl moiety, tetrazole substituted in the phenylmoiety, 3-nitro-1,2,4-triazole substituted in the phenyl moiety,thiazole substituted in the phenyl moiety, pyrrole substituted in thephenyl moiety, benzotriazole substituted in the phenyl moiety, andbenzohydroxytriazole substituted in the phenyl moiety.

PNA-DNA chimeras can be assembled by a variety of methods. Probably themost efficient method will use currently available techniques andinstrumentation available for DNA synthesis as previously described. Inparticular, nucleic acids (DNA and RNA) are now routinely synthesizedusing automated machines, numerous synthesis supports and variousprotection chemistries. The following U.S. patents cover a broad rangeof differing supports and protection chemistries and are hereinincorporated by reference. See U.S. Pat. Nos. 5,262,530; 4,415,732;4,458,066; 4,725,677 (RE 34,069); and 4,923,901. Automated equipment andreagents are commercially available from PerSeptive Biosystems, PerkinElmer (Applied Biosystems Division) and Pharmacia. Special 5′-aminosynthons are described in Smith et al., Nucleic Acids Res. (1985)13:2399 and in Sproat et al., Nucleic Acids Res. (1987) 15:6181. Special5′-thio synthons are described in Sproat et al., Nucleic Acids Res.(1987) 15:4837. The reagents described in the above references aresuitable for use on standard DNA synthesis instruments.

The preferred commercial method for nucleic acid synthesis utilizes theabove reagents and methods as generally described by Koester et al. inU.S. Pat. No. 4,725,677 (RE 34,069). Consequently, the preferredsynthons are cyanoethyl phosphoramidites having acid labile protectionof the backbone 5′ hydroxyl group and base labile acyl-type protectionof the exocyclic amino groups of the nucleobases.

The preferred acid labile backbone protecting group is4,4′-dimethoxytriphenylmethyl (DMT). DMT is typically chosen because itcan be removed farily rapidly (1-3 mintues) during each synthetic cyclewith solutions containing 1-4% dichloroacetic acid or trichloroaceticacid in dichloromethane. Protecting groups with increased acid labilitycompared to DMT are susceptible to premature deprotection during theacid catalyzed coupling reactions (tetrazole is typically the acidicspecies). Protecting groups with decreased acid lability compared to DMTrequire longer reaction times and/or harsher reaction conditions forcomplete removal. Generally, harsher acidic deprotection conditions areavoided since the purine nucleobases are particularly susceptible todecomposition in acid. Although the aforementioned problems withprotecting groups and synthetic conditions may be minimal during eachsynthetic cycle, the cumulative effect can generate significantimpurities in oligonucleotide synthesis. Accordingly, as the length ofthe oligonucleotide increases, its purity tends to decrease.

Generally, base labile protecting groups are utilized for protection ofthe exocyclic amino groups of the nucleobases so that an orthogonallyprotected nucleic acid synthon results. The base labile protectinggroups typically remain a part of the growing nucleic acid chain, thenare removed simultaneously with the cleavage of synthesized nucleic acidfrom the solid support. A concentrated ammonium hydroxide solution isoften used for the “deprotection and cleavage step.” Koester et al. usedbase labile acyl-type protecting groups which are usually treated for6-24 hours at elevated temperature (about 55° C.) for complete removalof these nucleobase protecting groups. Other protecting groups have beendeveloped which are removed under the same conditions but in less time(from about 15-60 minutes). Examples of these improved protecting groupsinclude phenoxyacetyl, t-butyl phenoxyacetyl and amindine-typeprotecting groups. While these protecting groups have increased baselability, typically only the time necessary for removal is reduced.

In addition, the PNA synthons can be coupled to each other to form a PNAoligomer (PNA). Because the chemistry of the PNA synthons is compatiblewith commercially available synthesizers, the synthons are readilytransformed into polymeric chains of various lengths and sequences.

Generally, before removal of the nucleobase protecting groups, the PNAoligomer will have the formula:

KLQMN

The letters K and N represent chemical bonds. The letter Q is a chemicalbond or a linker as previously described. Each of L and M is the same ordifferent and is independently a PNA moiety having the formula:

The entities G, Ba, R⁷, g, h and j are as previously described.

The oligomer can be assembled using standard peptide couplingprocedures. Following completion of the desired PNA sequence, removal ofall protecting groups, and cleavage from the solid support, ifnecessary, will produce the PNA molecule. Proper choice of the supportis important to avoid harsh acid treatments and preferrably will allowcleavage and deprotection to occur in basic solutions. Methods forpreparing suitable membrane supports are found in U.S. Pat. No.4,923,901. In addition, preferrably the support will be functionalizedwith hydroxyl groups to which the first PNA synthons is attached as anester.

Various methods already described in the chemical literature for peptidesynthesis are generally applicable to PNA oligomer synthesis. Thesemethods include, but are not limited to, solid phase peptide synthesisand solution synthesis. For example, in solid-phase synthesis, followingcoupling of the first amino acid, the next step is the systematicelaboration of the desired PNA chain. This elaboration involves repeateddeprotection/coupling cycles. The temporary backbone protecting group onthe last-coupled amino acid, such as Fmoc, is quantitatively removed bya suitable treatment, for example by base treatment with piperidine, soas to liberate the N-terminal amine function.

The next desired N-protected amino acid is then coupled to theN-terminal of the last-coupled amino acid. This coupling of theC-terminal of an amino acid with the N-terminal of the last-coupledamino acid can be achieved in several ways. For example, the carboxylgroup of the incoming amino acid can be reacted directly with theN-terminal of the last-coupled amino acid with the assistance of acondensation reagent such as, for example, dicyclohexylcarbodiimide(DCC) (Sheehan & Hess, et al., J. Am. Chem. Soc., 1955, 77, 1067) anddiisoproplycarbodiimide (DIC) (Sraantakis et al., Biochem. Biophys. Res.Commun., 1976, 73, 336) or derivatives thereof. Alternatively, it can bebound by providing the incoming amino acid in a form with the carboxylgroup activated by any of several methods, including the initialformation of an active ester derivative such as a 2,4,5-trichlorophenylester (Pless, et al., Helv. Chim. Acta, 1963, 46, 1609), a phthalimidoester (Nefkens, et al., J. Am. Chem. Soc., 1961, 83, 1263), apentachlorophenyl ester (Kupryszewski, Rocz. Chem., 1961, 35, 595), apentafluorophenyl ester (Kovacs, et al., J. Am. Chem. Soc., 1963, 85,183), an o-nitrophenyl ester (Bodanzsky, Nature, 1955, 175, 685), animidazole ester (Li, et al., J. Am. Chem. Soc., 1970, 92, 7608), and a3-hydroxy-4-oxo-3,4-dihydroquinazoline (Dhbt-OH) ester (Konig, et al.,Chem. Ber., 1973, 103, 2024 and 2034), or the initial formation of ananhydride such as a symmetrical anhydride (Wieland, et al., Angew.Chem., Int. Ed. Engl., 1971, 10, 336). BenzotriazolylN-oxytrisdimethylaminophosphonium hexafluorophospha te (BOP), “Castro'sreagent” (see, e.g., Rivaille, et al., Tetrahedron 1980, 36, 3413) isrecommended when assembling PNA molecules containing secondary aminogroups. Preferred reagents for activity the carboxylic acid groupsinclude 1-hydroxy-7-azabenzotriazole (HOAT) and its phosphonium anduronium salts. See Carpino, L. J. Am. Chem. Soc., (1993) 115, 4397.Finally, activated PNA monomers analogous to the recently-reported aminoacid fluorides (Carpino, J. Am. Chem. Soc., 1990,112, 9651) holdconsiderable promise to be used in PNA synthesis as well.

Following assembly of the desired PNA chain, including protectinggroups, the next step will normally be deprotection of the amino acidmoieties of the PNA chain and cleavage of the synthesized PNA from thesolid support. These processes can take place substantiallysimultaneously, thereby providing the free PNA molecule in the desiredform. Alternatively, in cases in which condensation of two separatelysynthesized PNA chains is to be carried out, it is possible by choosinga suitable spacer group at the start of the synthesis to cleave thedesired PNA chains from their respective solid supports (both peptidechains still incorporating their side-chain protecting groups) andfinally removing the side-chain protecting groups after, for example,coupling the two side-chain protected peptide chains to form a longerPNA chain.

After the PNA-DNA chimera has been synthesized and isolated, manypotential uses of the various sequence combinations are realized. Onesuch use is as a therapeutic or antisense agent. PNA-DNA chimeras can beadministered to organisms in a variety of ways or forms known in theindustry thereby assisting in the maintenance of a healthy organism orhelping increase the rate of recovery from poor health.

PNA-DNA chimeras may be useful as primers in a polymerase reaction. ThePNA-DNA chimera is contacted with a nucleic acid polymer to which thechimera recognizes and binds to, thereby beginning the polymerasereaction.

PNA-DNA chimeras also may be useful as probes for the detection of agenetic sequence.

The invention, being generally described above, is now more specificallyillustrated by way of the following examples, which are not meant tolimit the invention, unless otherwise noted.

EXAMPLES Example 1

Synthesis of 4,4′-dimethylbenzhydrol (I)

To 4.73 mole of magnesium chips was added dropwise, at a rate necessaryto maintain a vigorous reflux after initiation of the reaction, asolution containing 4.4 mole of 4bromo toluene which had been diluted toa total of 3 liters (L) with dry tetrahydrofuran (THF). After theaddition was completed, the reaction was allowed to stir until it hadcooled near room temperature (about one hour). To the prepared grignardreagent was added dropwise 4.3 mole of p-tolualdehyde at a ratenecessary to cause the reaction to reflux. After addition was completed,the reaction was allowed to stir until it had cooled near roomtemperature (about 30 minutes). The reaction was then concentrated toabout half volume and poured into a heterogeneous solution (brisklystirring) containing 2.5 liters of dichloromethane and a solutioncontaining 4.8 moles of potassium hydrogen sulfate diluted to 4.5 literswith water. After addition, a solution containing 3N HCl was added untilall the salts had dissolved (dichloromethane layer on top). The layerswere separated and the organic layer washed one time with dilute sodiumphosphate buffer (pH 5). The product was dried, filtered and evaporated.Yield: 970 grams green oil. The product was recrystallized from 1.6liters of hexane/ethylacetate (9:1). Yield: 660.1 grams (g) white solid(72%). A a second crop was obtained from the mother liquor byrecrystallizing from 500 mL ethylacetate/hexane (9:1). Yield: 108.0 gwhite solid (11.8%). Total Yield: 768.1 g, 3.62 mole (84%).

¹H-NMR deutero chloroform (CDCl₃): δ=7.3-7.0 (dd, 8H), 5.7 (s, 1H), 2.3(s, 6H), 2.2 (s, 1H)

Example 2

Synthesis of N-(4,4′-dimethylbenzhydroloxycarbonyl)-ethylenediamine (II)

To 130 millimole (mmole) of carbonyldiimidazole (CDI) suspended in 100mL of dichloromethane (DCM) and stirring at 0° C. was added dropwise asolution of 125 mmole of 4,4′-dimethylbenzhydrol dissolved in 60 mL ofdichloromethane. The reaction was allowed to stir for 30 minutes afterthe addition was complete. Thin layer chromatography (tlc) analysisafter 30 minutes indicated a complete reaction. The product wastransferred to a separatory funnel and washed two times with 70 mL ofwater. The dichloromethane layer was then dried with sodium sulfate,filtered, and evaporated. Yield: 40 g white solid.

To one mole of ethylenediamine stirring at 0° C. was added dropwise asolution containing the 40 g of isolated product dissolved in 150 mL ofdichloromethane. The reaction was then allowed to stir for 30 minutesafter the addition was completed. The solution was then transferred to aseparatory funnel and extracted four times with 100 mL of water. (Thelast wash contained a small amount of brine to minimize emulsionformation.) The dichloromethane layer was then dried with sodiumsulfate, filtered and evaporated. Yield: 37.8 g clear yellow oil (101%)

¹H-NMR (CDCl₃): δ=7.3-7.0 (dd, 8H), 6.7 (s, 1H), 5.5 (m, 1H), 3.2-3.1(dd, 2H), 2.8-2.7 (t, 2H), 2.3 (s, 6H), 1.1 (s, 2H)

Example 3

Synthesis ofN-(4.4′-dimethylbenzhydroloxycarbonyl)-N′-trifluoroacetylethylenediamine(III)

To the 37.8 g of N-(4,4′-dimethylbenzhydroloxycarbonyl)-ethylenediamineisolated in example 2 was added 200 mL of dichloromethane and 50 mmoleof triethylamine. The solution was cooled in an ice bath and then 150mmole of ethyl trifluoroacetate was added dropwise. A white solidproduct crystallized over the next one hour period and was thencollected by vacuum filtration. The filtrate was allowed to stirovernight and then transferred to a separatory funnel. After transfer,the solution was washed two times with sodium phosphate buffer (pH 6),dried with sodium sulfate, filtered and evaporated. The residue wasrecrystallized from 250 mL of hexane/ethylacetate (3:2). Yield: 16.6 gwhite solid (34%). The product initially collected by vacuum filtrationweighed 21.05 g (42%) Total Yield is 37.65 g (76%).

¹H-NMR (CDCl₃): δ=7.6 (m, 1H), 7.3-7.0 (dd, 8H), 6.7 (s, 1H), 5.4 (m,1H), 3.4-3.2 (m, 4H), 2.3 (s, 6H).

Example 4

Synthesis ofN-[N′-4,4′-dimethylbenzhydroloxycarbonyl-(2′-aminoethyl)]glycine (IV)

To 40 mmole ofN-(4,4′-dimethylbenzhydroloxycarbonyl)-N′-trifluoroacetyl-ethylenediaminestirring in 160 mL of dry tetrahydrofuran (THF) at 0° C. was added 44mmole of sodium hydride. The reaction was allowed to stir in an ice bathuntil all gas evolution had ceased and the deep blue color disappeared.To the reaction was then added 48 mmole of ethyl bromoacetate. Thereaction was then allowed to stir overnight while warming to roomtemperature. In the morning the solvent was evaporated and the residuerepartitioned with 100 mL of ethylacetate and 100 mL of sodium phosphatebuffer (pH 6). The organic layer was then washed one time with water andthen dried with sodium sulfate, filtered and evaporated. Yield: 19.72 gorange oil (90%).

The residue was dissolved in 240 mL of ethanol/acetonitrile (1:1) andthen 60 mL of water was added. The solution was cooled in an ice bathand then 160 mL of 2.5N aqueous lithium hydroxide was added. Reactionwas allowed to stir for 1 hour (hr) and then 3 N HCl was added dropwisevery slowly until the pH was 8-9 by paper. To the solution was thenadded 15 mL of 1M NaH₂PO₄ to adjust the pH to 7. A white precipitatethen formed and the solution was then concentrated on a rotoevaporateuntil approximately 150 mL of solvent was removed. The residue was thenextracted with 3×100 mL of ethylacetate. All ethylacetate layers werecombined and back extracted one time with IM phosphate buffer (pH 7).The ethylacetate layers were dried in sodium sulfate, filtered andevaporated. Yield: 14.3 g yellow foam. Recrystallized from 400 mLacetonitrile. Yield: 8:35 g (59%). Recrystallized again from 100 mL ACN.Yield: 7.63 g (54%).

¹H-NMR deutero methyl sulfoxide (d₆DMSO): δ=7.7 (m, 1H), 7.3-7.0 (dd,8H), 6.6 (s, 1H), 3.3-3.1 (m, 4H), 2.9 (m, 2H), 2.3 (s, 6H)

Example 5

Synthesis of 2-(1′-thyminyl)acetic acid

To 300 grams of thymine was added 750 mL hexamethyldisilazane and 15grams of ammonium sulfate. The stirring reaction was heated at refluxuntil no more gas was evolved. The reaction was then cooled to 80° C.and then 160 mL of ethylbromoacetate was added. Gentle heating of thereaction was continued until tlc analysis indicated that 95% of thestarting material was consumed. The reaction was then cooled to 20° C.in an ice bath and then 150 mL of methanol was added dropwise whilestirring in the ice bath. Once the addition of methanol was complete,2.5 liters of 3N NaOH was added carefully. The ice bath was then removedand gas was passed over the reaction rapidly to vaporize excess silanes.Then 80 grams of sodium hydroxide was added and the reaction was allowedto stir overnight. In the morning, 1 L of 6N HCl was added to thereaction to cause the product to crystallize. The product was thencollected by vacuum filtration and washed with dilute aqueoushydrochloric acid. The collected product was then suspended in 1.5 L ofacetonitrile and the solution allowed to reflux with stirring. Aftercooling overnight, the solid was again collected by vacuum filtration,washed with acetonitrile, dichloromethane, and acetonitrile. Yield: 327grams (75%).

¹H-NMR (d₆DMSO): δ=12.06 (s, 1H), 11,31 (s, 1H), 7.48 (s, 1H), 4.35 (s,2H), 1.74 (s, 3H)

Example 6

Synthesis of 2,2-dimethyl-1-cyanoethoxycarbonyl-imidazole (XIV)

To 250 mmole of carbonyldiimidazole suspended in 100 mL ofdichloromethane was added 260 mmole of 2-hydroxy-2-methyl-butyronitrile.The reaction stirred for about 20 hours and then was transferred to aseparatory funnel. An additional 250 mL of dichloromethane was added andthe solution was then washed three times with 100 mL of water. Theorganic layer was then dried with sodium sulfate, filtered and thenconcentrated to about 50 mL. Immediately thereafter, 200 mL of ether wasadded with brisk stirring and the solution was then cooled in an icebath for 30 minutes. The white solid product was then collected byvacuum filtration. Yield: 29.6 grams (61%).

¹H-NMR (d₆DMSO): δ=8.1 (m, 1H), 7.4 (m, 1H), 7.1 (m, 1H), 3.1 (s, 2H),1.8 (s, 6H)

Example 7

Synthesis of t-Butyl 2-(1′-cytosyl)acetate (V)

To 200 mmole of cytosine was added 250 mL of DMF and 215 mmoles ofpotassium-t-butoxide. The reaction was then heated to 60° C. with briskstirring and then immediately cooled to 0° C. To the ice cold solutionwas added dropwise 215 mmoles of t-butyl-bromoacetate. The reaction asallowed to stir one hour at 0° C. and then it was concentrated. Theresidue was poured into 500 mL of water containing 30 mmoles of 3Nhydrochloric acid. The solution stirred for 20 minutes and then thewhite solid product was collected by vacuum filtration. Yield: 32.08 g(71%).

¹H-NMR (d₆DMSO): δ=7.5 (d, 1H), 7.1 (s, 2H), 5.7 (d, 1H), 4.3 (s, 2H),1.4 (s, 9H)

Example 8

Synthesis of t-Butyl 2-[N′⁴-2,2-dimethyl-1-cyanoethoxycarbonyl(1′-cytosyl)]acetate (VI)

To 52 mmoles of 2,2-dimethyl-1-cyanoethoxycarbonyl-imidazole in 150 mLof dichloromethane (at 0° C.) was added 50 mmole of Methyltrifluoromethanesulfonate (dropwise). The reaction was allowed to stirfor 30 minutes and then 35 mmole of t-Butyl 2-(1′-cytosyl)acetate wasadded. The reaction was allowed to stir until complete by tlc analysis(24 hours). The product was transferred to a separatory funnel andextracted once with 50 mL of 5% potassium hydrogen sulfate and once with5% sodium bicarbonate. The organic layer was then dried with sodiumsulfate, filtered and evaporated. The residue was recrystallized withethylacetate. Yield: 7.35 g (60%).

¹H-NMR (CDCl₃): δ=7.5 (d, 1H), 7.1 (d, 1H), 4.5 (s, 2H), 2.9 (s, 2H),1.7 (s, 6H) 1.5 (s, 9H)

Example 9

Synthesis of2-[N′⁴-2,2-dimethyl-1-cyanoethoxycarbonyl(1′-cytosyl)]acetic acid (VII)

To 25 mmole of t-Butyl 2-[N′⁴-2,2-dimethyl-1-cyanoethoxycarbonyl(1′-cytosyl)]acetate was added 25 mL of dichloromethane and 50 mL oftrifluoroacetic acid (TFA). The reaction as allowed to stir until theester completely hydrolyzed (four hours). The solution was thenconcentrated and redissolved in 10 mL of dichloromethane. The productwas precipitated by adding this solution dropwise to 350 mL of brisklystirring ice cold ethyl ether. The product was then collected by vacuumfiltration. Yield: 7.60 g white solid (103%). [Does not appear to existas a TFA salt.]

¹H-NMR (d₆DMSO): δ=8.0 (d, 1H), 6.9 (d, 1H), 4.5 (s, 2H), 3.2 (s, 2H),1.5 (6H)

Example 10

Synthesis of t-Butyl 2-(9′-adenyl)acetate (VII)

To 200 mmoles of adenine was added 400 mL of dried dimethylformamide(DMF). To the stirring suspension was then added 230 mmoles of sodiumhydride. The solution was allowed to stir overnight at room temperatureand then cooled in an ice bath for 30 minutes. Thereafter, 220 mmoles oft-butyl bromoacetate was added dropwise. The solution as then stirredfor 30 minutes and then concentrated to about 50 mL under reducedpressure. The residue was then poured into 1.5 L of water containing 20mL of 10% sodium carbonate. After stirring 30 minutes, the white solidproduct which precipitated was collected by vacuum filtration. The wetproduct was then recrystallized from 300 mL of acetonitrile/water (9:1).The product crystals were collected by vacuum filtration. Yield: 25.94grams (52%).

¹H-NMR (d₆DMSO): δ=8.13 (s, 1H), 8.10 (s, 1H), 7.24 (s, 2H), 4.9 (s,2H), 1.4 (s, 9H)

Example 11

Synthesis of t-Butyl 2-[N′⁶-2,2-dimethyl-1-cyanoethoxycarbonyl(9′-adenyl)]acetate (IX)

To 2,2-dimethyl-1-cyanoethyoxycarbonyl-imidazole was added 100 mL ofdichloromethane. The solution was cooled in an ice bath for 30 minutesand then 50 mmole of Methyl trifluoromethanesulfonate was addeddropwise. The reaction was allowed to stir 30 minutes in an ice bath andthen 35 mmoles of t-Butyl 2-(9′-adenyl)acetate was added and thereaction stirred until complete by tlc analysis (four days). Thesolution was then transferred to a separatory funnel, extracted oncewith 70 mL of 5% potassium hydrogen sulfate, once with 5% sodiumbicarbonate, and once with a dilute sodium chloride solution. Theorganic layer was then dried with sodium sulfate, filtered andevaporated. The white solid product was crystallized from 130 mL ofethyl acetate. Yield: 10.17 gram (77%).

¹H-NMR (d₆DMSO): δ=10.5 (s, 1H), 8.6 (s, 1H), 8.4 (s, 1H), 5.1 (s, 2H),3.2 (s, 2H), 1.6 (s, 6H), 1.4 (s, 9H)

Example 12

Synthesis of 2-[N′6-2,2-dimethyl-1-cyanoethoxycarbonyl(9′-adenyl)]aceticacid (X)

To 28 mmole of t-Butyl 2-[N′⁶-2,2-dimethyl-1-cyanoethoxycarbonyl(9′-adenyl)]acetate was added 25 mL of dichloromethane and 50 mL oftrichloroacetic acid (TFA). The reaction was stirred until all the esterwas hydrolyzed as indicated by tlc analysis (4 hours). The solution wasthen concentrated to an oil and the residue redissolved in 10 mL ofdichloromethane. The product was then precipitated by dropwise additionof this solution to briskly stirring ice cold ethyl ether. The productwas then collected as a white solid by vacuum filtration. Yield: 11.55 goff-white solid (95%). [Product believed to exist as a TFA salt.]

¹H-NMR (d₆DMSO): δ=8.6 (s, 1H), 8.5 (s, 1H), 5.1 (s, 2H), 3.2 (s, 2H),1.6 (s, 6H)

Example 13

Synthesis of Benzyl 2-[6′-chloro(9′-purinyl)]acetate (XI)

To 6-chloro-2-amino purine (300 g. 1.77 mole; Pharma-Waldorf GmbH,Germany, P/N 471720) and potassium carbonate (366 g; 2.65 mole, AldrichChemical, Milwaukee, Wis. (hereinafter Aldrich) P/N 34,782-5) was addeddimethyl formamide (DMF, 3 L) and the solution was warmed until all the2-amino-6-chloropurine dissolved (84° C.). The mixture was then cooledin an ice bath and benzyl-2-bromoacetate (299 mL, 1.89 mole; Aldrich P/N24,563-1) was added dropwise over the course of one and one half hours.The mixture was stirred for an additional three hours at 0° C. and wasthen stirred overnight at ambient temperature. The following day thereaction mixture was filtered and the filtrate was then poured into asolution containing 7 liters of water and 150 mL of concentratedhydrochloric acid (HCl). The mixture was stirred for 2 hr. and theproduct was then isolated by filtration. The product was washedthoroughly with water and subsequently recrystallized by portion-wiseaddition of the solid to boiling acetonitrile (3 L). The very redsolution was left overnight and filtered the next day. The product waswashed thoroughly with methanol and then diethylether. Yield 386 g (69%)

¹H-NMR (d₆ DMSO) δ=8.14 (1H, s), 7.4-7.3 (5 H, m), 7.02 (2 H, s), 5.21(2 H, s), 5.08 (2H, s).

Example 14

Synthesis of Benzyl2-[N′²-2-(Methylthio)ethoxycarbonyl-6′-chloro(9′-purinyl)]acetate (XII)

To 50 mmol of Benzyl 2-[6′-chloro(9′-purinyl)]acetate was added about200 mL of freshly distilled tetrahydrofuran. The reaction was cooled for20 minutes in an ice bath and then 20 mmol of triphosgene was added. Thereaction was allowed to stir 30 minutes at 0° C. and then 130 mmol ofdiisopropylethylamine was added dropwise. After stirring 20 minutes at0° C., 70 mmol of 2-(methylthio)-ethanol was added. The reaction wasallowed to stir overnight while warming to room temperature. In themorning, the reaction was concentrated to about half volume and thenpoured into a stirring solution containing 500 mL of water and 30 mmolof HCl. This mixture was allowed to stir for 30 minutes and then theproduct was then collected by vacuum filtration. The product wasrecrystallized from ethanol. Yield 74%

¹(d₆DMSO) δ=10.8 (1H, s), 8.5 (1H, s), 7.35 (5H, m), 5.22 (4H, m), 4.25(2H, t), 2.75 (2H, t), 2.15 (3H, s)

Example 15

Synthesis of 2-[N′²-2-(Methylthio)ethoxycarbonyl(9′-guanyl)]acetic acid(XIII)

To 75 mmol of 95% sodium hydride was added about 100 mL of freshlydistilled tetrahydrofuran. The solution was cooled in an ice bath for 20minutes and then 75 mmol of 3-hydroxypropionitrile was added. Reactionwas stirred at 0° C. for 2 hours and then 15 mmol of Benzyl2-[N′²-2-(Methylthio)-ethoxycarbonyl-6′-chloro(9′-purinyl)]acetate wasadded. The reaction was allowed to stir overnight while warming to roomtemperature. In the morning, the solvent was completely evaporated andthen a solution containing 200 mL of water, 54 grams of sodium chlorideand 8 grams of K₂S₂O₇ was added. The solution was stirred briskly for 15minutes and then the solid product filtered off. The product as purifiedby boiling in acetonitrile. Yield 83%.

¹HNMR (d₆DMSO) δ=11.52 (1H, s), 11.37 (1H, s) 7.93 (1H, s), 4.9 (2H, s),4.35 (2H, t), 2.785 (2H, t), 2.15 (3H, s)

Example 16

Synthesis ofN-[N″-4,4′-dimethylbenzhydroloxycarbonyl-(2″-aminoethyl)]-N-[2-(1′-thyminyl)acetyl]glycine(XIX)

To 3 mmole of N-[N′-,4,4′-dimethylbenzhydroloxycarbonyl-(2′-aminoethyl)]glycine was added 15 mL of acetonitrile (ACN) and 12mmole of N-methylmorpholine (NMAM). The solution was allowed to cool inan ice bath and then 6 mmole of triisopropylsilyl chloride was added.The solution was allowed to stir for two hours at 0° C. and then it wasadded to the product of the following reaction as described below.

To 3.3 mmole of 2-(1′-thymiinyl)acetic acid was added 15 mL ofacetonitrile and 12 mmole of N-methylmorpholine. The solution wasallowed to stir while cooling to 0° C. and then 3.7 mmole oftrimethylacetyl chloride was added to the solution. After stirring for15 minutes aliquots of reaction were removed and reacted withdiethylamine to determine whether complete anhydride formation hadoccurred. After tlc analysis had indicated completion formation of theanhydride, the above described reaction was added dropwise to theproduct of this reaction and this mixture was then allowed to stir for30 minutes. The product was then evaporated and the residue partitionedwith 30 mL of ethyl ether and 30 mL of a solution containing 3%N-methylmorpholine in water.

The layers were separated, and the organic layer washed one time with 30mL of a solution containing 3% N-methylmorpholine in water. The organiclayer was dried with sodium sulfate and filtered into a second flask.The total volume of the solution was adjusted to 75 mL by the additionof more ethyl ether. To this solution was added 12 mmole ofN-methylmorpholine and 3 mmole of triethylamine trihydrofluoride(Et₃N.3HF). To the solution was added 30 mL of water after 5 min (pH to7-8 by paper). To the solution was added additional ethyl ether and 30mL of a solution containing 3% N-methylmorpholine in water. The layerswere separated and the aqueous layer was evaporated. The residue wasthen partitioned with 30 mL of dichloromethane and 40 mL of a pH 3.5*buffer. The layers were separated and the organic layer was then driedwith sodium sulfate, filtered and 6 mmole N-methyl morpholine was addedto the filtrate. The filtrate was then evaporated and the residuedissolved in a minimal amount of dichloromethane. The product was thenprecipitated by dropping this solution into briskly stirring ice coldethyl ether. The product was collected by vacuum filtration. Yield:0.872 g (48%).

*[pH 3.5 buffer consisted of 0.2 M citric acid, 0.2M Na₂HPO₄, 0.2MNaH₂PO₄]

¹H-NMR (d₆DMSO): δ=11.3 (m, 1H,), 7.7-7.4 (mm, 1H), 7.3-7.0 (m, 9H), 6.6(m, 1H), 4.8 (s, mj, 2H), 4.4 (s, mi, 2H), 4.0 (s, mi, 2H), 3.9 (s, mj,2H), 3.6 (t, ½ NMM), 3.5-3.0 (m, 4H), 2.4 (t, ½ NMM), 2.25 (s, 6H), 2.2(s, ½ NMM), 1.7 (s, 3H) The PNA synthons prepared produced rotomers dueto hindered rotation about the amide bond. When signals appear for eachrotomer form, they are designated mj for major signal (the largersignal) and mi for minor signal. The number of protons designatedrepresents the total number for the combined signals. For this monomerthe abundance of rotomer is approximately equal and therefore thedesignation is merely inserted for convenience of the reader.

Example 17

Synthesis ofN-[N′-4,4′-dimethylbenzhydroloxycarbonyl-(2″-aminoethyl)]-N-[2-[N′⁴-2,2-dimethyl-1-cyanoethoxycarbonyl(1′-cytosyl)]acetyl]glycine(XVI)

To 3 mmole ofN-[N′-4,4′-dimethylbenzhydroloxycarbonyl-(2′-aminoethyl)]glycine wasadded 15 mL of acetonitrile and 9 mmole of N-methylmorpholine. Thesolution was allowed to cool in an ice bath and then 6 mmole oftriisopropylsilyl chloride was added. The solution was allowed to stirfor two hours at 0° C. and then it was added to the product of thefollowing reaction as described below.

To 3.3 mmole of2-[N′⁴-2,2-dimethyl-1-cyanoethoxycarbonyl(1′-cytosyl)]acetic acid wasadded 15 mL of acetonitrile and 9 mmole of N-methylmorpholine. Thesolution was allowed to stir while cooling to 0° C. and then 3.7 mmoleof trimethylacetyl chloride was added to the solution. After stirringfor 15 minutes aliquots of reaction were removed and reacted withdiethylamine to determine whether complete anhydride formation hadoccurred. After tlc analysis had indicated complete formation of theanhydride, the above described reaction was added dropwise to theproduct of this reaction and this mixture was then allowed to stir for30 minutes.

The product was then evaporated and the residue partitioned with 30 mLof ether and 30 mL of a solution containing 3% N-methylmorpholine inwater. The layers were separated, and the organic layer washed one timewith 30 mL of a solution containing 3% N-methylmorpholine in water. Theorganic layer was dried with sodium sulfate and filtered into a secondflask. To this solution was added 9 mmole of N-methylmorpholine and 2.2mmole of triethylamine trihydrofluoride. Because the pH was determinedto be very high, excess triethyamine trihydrofluoride was added toadjust the pH to 7 or 8 by paper. To the solution was added additionalethyl ether and 30 mL of a solution containing 3% N-methylmorpholine inwater. The layers were separated and the product determined to be in theaqueous layer. The organic er was washed one time with 30 mL of 3%N-methylmorpholine in water. The water layers were combined and thenevaporated to a white gel. The gel was co evaporated two times fromwater and one time from dry tetrahydrofuran (THF). The residue was thenpartitioned with 25 mL of dichloromethane and 30 mL of a pH 3.5* buffer.Th-e product was determined to be in the dichloromethane layer. Thelayers were separated and the organic layer washed one time with dilutesodium chloride solution. The organic layer was then dried with sodiumsulfate, filtered and evaporated. Yield: 1.61 grams yellow foam (73%).The foam was dissolved in a minimal amount of dichloromethane and theproduct precipitated by dropping this solution into a stirring solutionof ice cold ethyl ether. Yield: 1.06 g (48%)

¹H-NMR (d₆DMSO): δ=7.9-7.7 (m, 1H), 7.6 (m, mj, 1H), 7.4 (m, mi, 1H),7.3-7.0 (mn, 9H), 7.0-6.8 (m, 1H), 6.6 (m, 1H), 4.8 (s, mj, 2H), 4.6 (s,mi, 2H), 4.2 (s, mi, 2H), 4.0 (s, mj, 2H), 3.6 (t, ½ NMM), 3.5-3.0 (m,4H), 2.4 (t, ½ NMM), 2.25 (s, 6H), 2.2 (s, ½ NMM), 1.5 (s, 6H)

*[PH 3.5 buffer consisted of 0.2 M citric acid, 0.2M Na₂HPO₄, 0.2MNaH₂PO₄]

Example 18

Synthesis ofN-[N″-4,4′-dimethylbenzhydroloxycarbonyl-(2″-aminoethyl)]-N-[2-[N′⁶-2,2dimethyl-1-cyanoethoxycarbonyl(9′-adenyl)]acetl]glycine(XVII)

To 6 mmole of N-[N′-,4,4′-dimethylbenzhydroloxycarbonyl-(2′-aminoethyl)]glycine was added 35 mL of acetonitrile and 24 mmole ofN-methylmorpholine. The solution was allowed to cool in an ice bath andthen 6 mmole of triisopropylsilyl chloride was added. An additional 3mmole of triisopropylsilyl chloride was added because the tlc analysisof the ester formation indicated incomplete reaction. The solution wasallowed to stir for a total of 1.5 hours at 0° C. and then was added tothe product of the following reaction as described below.

To 7 mmole of2-[N′⁶-2,2-dimethyl-1-cyanoethoxycarbonyl(9′-adenyl)]acetic acid wasadded 30 mL of acetonitrile and 28 mmole of N-methylmorpholine. Thesolution was allowed to stir while cooling to 0° C. and then 7.7 mmoleof trimethylacetyl chloride was added to the solution. After stirringfor 20 minutes aliquots of reaction were removed and reacted withdiethylamine to determine whether complete anhydride formation hadoccurred. Because the anhydride did not appear to completely form, anadditional 0.7 mmole of trimethylacetyl chloride was added and thereaction was stirred for an additional 10 min. After tlc analysis hadindicated completion formation of the anhydride, the above describedreaction was added dropwise to the anhydride product of this reactionand this mixture was then allowed to stir for 30 minutes.

The product was then evaporated and the residue partitioned with 60 mLof ethyl ether and 60 mL of a solution containing 3% N-methylmorpholinein water. The layers were separated, and the organic layer washed onetime with 30 mL of a solution containing 3% N-methylmorpholine in water.The organic layer was dried with sodium sulfate and filtered into asecond flask. To this solution was added 24 mmole of N-methylmorpholineand 60 mmole of triethylamine trihydrofluoride. To the solution wasadded 60 mL of water and then the solutions were stirred untileverything, dissolved (pH at about 8 by paper). The layers wereseparated and the aqueous layer was evaporated. The residue was thenpartitioned with 60 mL of dichloromethane and 80 mL of a pH 3.5* buffer.The layers were separated and the organic layer was then dried withsodium sulfate, filtered and 12 mmole N-methyl morpholine was added tothe filtrate. The filtrate was then evaporated and the residue dissolvedin a minimal amount of dichloromethane. The product was thenprecipitated by dropping this solution into briskly stirring ice coldethyl ether. The product was collected by vacuum filtration. Yield: 2.58g (57%).

¹H-NMR (d₆DMSO): δ=10.5 (s, 1H), 8.5 (d, 1H), 8.3 (d, 1H), 7.7 (m, mj,1H), 7.4 (m, mi, 1H), 7.3-7.0 (m, 8H), 6.6 (m, 1H), 5.2 (s, mj, 2H), 5.1(s, mi, 2H), 4.2 (s, mi, 2H), 4.0 (s, mj, 2H), 3.6 (t, ½ NMM), 3.5-3.0(mn, 4H), 2.4 (t, ½ NMM), 2.25 (s, 6H), 2.2 (s, ½ NMM), 1.6 (s, 6H)

Example 19

Synthesis ofN-[N′-4,4′-dimethylbenzhydroloxycarbonyl-(2″-aminoethyl)]-N-[2-[N′-²-2-(Methylthio)ethoxycarbonyl(9′-guanyl)]acetyl]glycine(XVHII)

To 5 mmole of N-[N′-,4,4′-dimethylbenzhydroloxycarbonyl-(2′-aminoethyl)]glycine was added 25 mL of acetonitrile and 20 mmole ofN-methylmorpholine. The solution was allowed to cool in an ice bath andthen 10 mmole of triisopropylsilyl chloride was added. The solution wasallowed to stir for 1.5 hours at 0° C. and then was added to the productof the following reaction as described below.

To 6 mmole of 2-[N′²-2-(Methylthio)ethoxycarbonyl(9′-guanyl)]acetic acidwhich was co-evaporated 2× from 10 mL of dry DMF) was added 25 mL ofacetonitrile. The solution was allowed to stir while cooling to 0° C.over 20 minutes. Thereafter, 7 mmole of trimethylacetyl chloride wasadded. Next, 20 mmole of N-methylmorpholine was added dropwise and thesolution was allowed to stir for 20 minutes. (As usual, aliquots ofreaction were removed and reacted with diethylamine to determine whethercomplete anhydride formation had occurred.)

After tlc analysis had indicated complete formation of the anhydride,the above described reaction was added dropwise to the product of thisreaction and this mixture was then allowed to stir for 1 hr. The productwas then evaporated (not to dryness) and the residue partitioned with 50mL of ethyl ether and 50 mL of a solution containing 3%N-methylmorpholine in water. The layers were separated, and the organiclayer was increased in volume by 20 mL (ether added) and then it waswashed one time with 30 mL of a solution containing 3%N-methylmorpholine in water. The organic layer was dried with sodiumsulfate and filtered into a second flask. To this solution was added 20mmole of N-methylmorpholine and 5 mmole of triethylaminetrihydrofluoride. To the solution was added 50 mL of a solutioncontaining 3% N-methylmorpholine in water (pH at about 8 by paper). Thelayers were separated and the aqueous layer was concentrated to 5-10 mL.This was transferred to a separatory funnel and 50 mL of ethyl acetateand 75 mL of pH 3.5* buffer was added. The layers were separated and theorganic layer was then dried with sodium sulfate, filtered and 10 mmoleN-methyl morpholine was added to the filtrate. The filtrate was thenevaporated and the residue dissolved in a minimal amount ofdichloromethane. The product was then precipitated by dropping thissolution into 200 mL ice cold ethyl ether. The product was collected byvacuum filtration. Yield: 1.65 g (43%).

¹H-NMR (d₆DMSO): δ=11.4 (s, 1H), 7.8 (d, 1H), 7.6 (m, mj, 1H), 7.5 (m,mi, 1H), 7.3-6.9 (m, 9H), 6.6 (m, 1H), 5.0 (s, mj, 2H), 4.9 (s, mi, 2H),4.4-4.2 (m, 2H), 4.16 (s, mi, 2H), 4.0 (s, mj, 2H), 3.6 (t, ½ NMM),3.55-3.0 (m, 4H), 2.8-2.6 (m, 2H), 2.4 (t, ½ NMM), 2.25 (m, 6H), 2.2 (s,½ NMM), 2.1 (s, 3H)

Example 20

Synthesis ofN-[N″-4,4′-dimethylbenzhydroloxycarbonyl-(2⊖-aminoethyl)]-N-[2-[N′²-2-(Methylsulfonyl)ethoxycarbonyl(9′-guanyl)]acetyl]glycine

To 1 mmole of N-[N″-4,4′-dimethylbenzhydroloxycarbonyl-(2″-aminoethyl)]-N-[2-[N′²-2-(Methylthio)ethoxycarbonyl(9′-guanyl)]acetyl]glycinewas added 6 mL of water and 1 mmole of N-methylmorpholine. The solutionwas stirred until all the solid dissolved. Once dissolved, 1 mmolesodium tungstate was added to the stirring solution. Then 2.5 mmole ofhydrogen peroxide (3% hydrogen peroxide in water) was added. Thereaction as allowed to stir and was monitored by HPLC analysis. After anhour of stirring, the reaction was heated to 50° C. to increase the rateof oxidation. Because oxidation did not appreciably increase, thereaction was then cooled and an additional 2 mL of 3% hydrogen peroxidein water was added. Thereafter, 250 microliters (μL) of 3% hydrogenperoxide solution was added every five minutes until HPLC analysisindicated that only about 5% unoxidized material remained. The reactionwas allowed to stir for an additional 30 minutes and then transferred toa separatory funnel. To the reaction was added 15 mL of dichloromethaneand 15 mL of pH 3.5* buffer. Because an emulsion formed, to the solutionwas added 10 mL of brine and the solution was allowed to stand untilseparated. The organic layer was collected, dried with sodium sulfate,filtered and 100 μL of N-methylmorpholine was added to the filteredsolution. Because the additional of N-methylmorpholine gave aheterogeneous solution, the entire solution was returned to theseparatory funnel and 10 mL of water, 10 mL of brine, and 10 mLdichloromethane was added. The layers were separated and the organiclayer was collected, dried with sodium sulfate, filtered and 200 mL ofN-methylmorpholine added to the filtered product. The solution wasevaporated and the residue dissolved in a minimum amount ofdichloromethane. The product was then precipitated by dropwise additionof this solution to briskly stirring ice cold ethyl ether. The whitesolid product was collected by vacuum filtration. Yield: 0.561 g yellowsolid (75%).

¹H-NMR (d₆DMSO): δ=11.4 (s, 1H), 7.8 (d, 1H), 7.7-7.5 (m, 1H), 7.3-6.9(m, 9H), 6.6 (m, 1H), 5.0 (s, mi, 2H), 4.9 (s, mj, 2H), 4.6-4.4 (m, 2H),4.0 (s, mi, 2H), 3.9 (s, mj, 2H), 3.6 (t, ½ NMM), 3.55-3.2 (m, 6H), 3.2(s, 3H), 2.4 (t, ½ NMM), 2.25 (m, 6H), 2.2 (s, ½ NMM)

Example 21

Synthesis of PNA sequence: H₂N-CTTCTCC-CONH₂ (SEQ ID NO: 1)

Synthesis resin: 50 Milligrams of Boc-BHA-PEG-PS from PerSeptiveBiosystems (P/N GEN063050) with amino group loading of 0.145 mmole/gram.

Scale: 7.25 micromole (μM) scale synthesis

Other equivalents:

 5 equivalents synthon 36 μM  4 equivalents HATU^(!) 11 milligrams (mg)per coupling 10 equivalents N-methylmorpholine  8 μL per coupling^(!)HATU = O-7-azabenzotriazol-1-yl)-1,1,3,3,-tetramethyluroniumhexafluorophosphate

Monomer was prepared immediately before each coupling step. To the drypowder in a 1 mL centrifuge tube was added 180 μL of a solutioncontaining 0.4 M N-methylmorpholine in dimethylformamide(DMF)/dichloromethane (DCM) (1:1) to dissolve. To the solution was thenadded 180 μL of 0.16M HATU dissolved in DMF. The solution was then mixedand used immediately for coupling reactions. (Final monomerconcentration was approximately 0.1 M)

Synthetic Cycle Reaction Step Time No. Description Reagents (min) 1Deblock Terminal Trifluoroacetic Acid 2.5 Amino Protecting(TFA)/m-cresol (95:5) Group 2 Repeat step 1:1 time 3 Wash DMF/DCM (1:1)4 Neutralize Pyridine 5 Couple Activated Synthon 15 Coupling Mixtureprepared as described above 6 Wash DMF/DCM (1:1) 7 Perform Kaiser Teston 4 sample of resin 8 Capping 5% acetic anhydride/DMF 2.5 9 WashDMF/DCM (1:1) 10 Repeat steps 1-9 until polymer is assembled

Cleavage:

Removed 7.2 milligram (mg) of resin. To the resin, in an Ultrafreedevice (Millipore P/N SE3P230J3), was added 100 μL of tetrahydrofuranand 300 μL of concentrated ammonium hydroxide solution. The tube wassealed and the solution heated at 55° C. overnight. The solution wasremoved from the beads by carefully pipetting the beads to an Ultrafreedevice (Millipore P/N SE3P230J3) followed by centrifugation. The resinbeads were then washed four times by the addition of dried THF followedby centrifugation. Finally the resin was dried in vacuum.

The resin was again transferred to a Ultrafree device (Millipore P/NSE3P230J3) and then subjected to High Cleavage Acid^(#) for 1.5 hours.The acid cleavage solution was then removed by centrifugation and theresin washed two times with more High Cleavage Acid solution. To thecollected acid solution was then added 1 mL of ether and the centrifugetube was centrifuged until the PNA oligomer precipitated. The ether wasdecanted and the PNA pellet was washed two more times with ether bysuspension of the pellet followed by centrifugation to regenerate apellet. The residual ether was allowed to completely evaporate from thepellet and then the pellet was dissolved in 1 mL of a solution of 0.1%TFA in water. Reversed phase HPLC analysis of the crude product obtainedis represented in FIG. 9. Further, the product was the analyzed byMatrix Aassisted Laser Desoption Ionization-time of flight (MALDI-TOF)Mass Spectrometry on a prototype mass spectrometer. Calculated molecularweight was (M+H) 1822 amu, mass found was 1823 amu (within the error ofthe mass spec apparatus).

^(#)High Cleavage Acid=TFA/trifluoromethanesulfonic acid(TFMSA)/m-cresol (7:2:1)

Example 22

Synthesis of PNA Sequences H₂N-CGCTATACCC-CONH₂ (SEQ ID NO: 2)

Synthesis resin: 80 Milligrams of Boc-BHA-PEG-PS from PerSeptiveBiosystems (P/N GEN063050) with amino group loading of 0.145 mmole/gram.

Scale: 11.6 micromole (μM) scale synthesis

Other equivalents:

 5 equivalents synthon 58 μM  4 equivalents HATU 18 mg per coupling 10equivalents N-methylmorpholine  8 μL per coupling

Monomer was prepared immediately before each coupling step. To the drypowder in a 1 mL centrifuge tube was added 145 μL of a solutioncontaining 0.8 M N-methylmorpholine in DMF/DCM (2:1) to dissolve. To thesolution was then added 145 μL of 0.32 M HATU dissolved in DMF. Thesolution was then mixed and used immediately for coupling reactions.(Final monomer concentration was about 0.2 M)

Synthetic Cycle Reaction Step Time No. Description Reagents (min) 1Deblock Terminal Amino Trifluoroacetic Acid 1 Protecting Group(TFA)/m-cresol (95:5) 2 Repeat step 1:1 time 3 Wash DMF/DCM (1:1) 4Neutralize Pyridine 5 Couple Activated Synthon 10 Coupling Mixtureprepared as described above 6 Wash DMF/DCM (1:1) 7 Perform Kaiser Teston 4 sample of resin 8 Capping 5% acetic anhydride/DMF 2 9 Wash DMF/DCM(1:1) 10 Repeat steps 1-9 until polymer is assembled Notes: Kaiser testwas slightly positive at coupling #8 (cytosine synthon). Kaiser was verypositive at coupling #9 (guanine synthon). Had to double couple at step9. Let coupling reaction at coupling #10 (cytosine synthon) go 15minutes. Kaiser still positive. Performed double coupling at coupling#10. After final wash, the resin was dried in vacuum. Total weight:0.1087 g (weight increase 28.7 mg)

Cleavage:

Removed 7.2-7.5 milligram (mg) of resin. To the resin, in Ultrafreedevice (Millipore P/N SE3P230J3), was added 100 μL of tetrahydrofuranand 300 μl of concentrated ammonium hydroxide solution. The tube wassealed and the solution heated at 56° C. for 2 hours. The solution wasremoved from the beads by carefully pipetting the beads to a Ultrafreedevice (Millipore P/N SE3P230J3) followed by centrifugation. The resinbeads were then washed four times by the addition of dry THF followed bycentrifugation. Finally, the resin was dried in vacuum.

The resin was again transferred to a Ultrafree device (Millipore P/NSE3P230J3) and then subjected to High Cleavage Acid^(#) for 1 hour. Theacid cleavage solution was then removed by centrifugation and the resinwas again treated with High Cleavage Acid for 1 hour. The acid cleavagesolution was then removed by centrifugation whereby both acid cleavagesolutions were combined in the bottom of the centrifuge tube of theUltrafree device. To the collected acid solution was then added 1 mL ofether and the centrifuge tube was centrifuged until the PNA oligomerprecipitated. The ether was decanted and the PNA pellet was washed twomore times with ether by suspension of the pellet followed bycentrifugation to regenerate a pellet. The residual ether was allowed tocompletely evaporate from the pellet and then the pellet was dissolvedin 1 mL of a solution of 0.1% TFA in water. Reversed phase analysis ofthe crude oligomer is represented in FIG. 10. Further, the product wasthe analyzed by Matrix Assisted Laser Desoption Ionizaton-time of flight(MALD-TOF) Mass Spectrometry on a prototype mass spectrometer.Calculated molecular weight was 2648.5 amu, mass found was (M+H) 2650amu (within the error of the mass spectrometric apparatus).

^(#)High Cleavage Acid=TFA/trifluoromethanesulfonic acid(TFMSA)/m-cresol (7:2:1)

Example 23

Synthesis of PNA/DNA Chimera:

Chimera sequence synthesized: DMBhoc-HNCACAC-CONH-linlker-5′-CCAGT-3′-OH(SEQ ID NO: 3), wherein the underlined sequence represents the PNAsequence and the bold sequence represents the DNA sequence.

Procedure:

A support bound 5′-amine modified DNA oligomer having the sequenceMMT-amninohexyl-CCAGT was synthesized using a commercially availableEXPEDITE® DNA synthesizer (PerSeptive Biosystems) running a standardprotocol for a 0.2 μM scale synthesis. The synthesis support was an 0.2μM MEMSYN® synthesis device (P/N GEN050034) and the synthons werestandard phosphoramidites having acyl (benzoyl, isobutyryrl) exocyclicamino protecting groups. The final coupling was performed using acommercially available MMT-aminohexyl-phosphorarnidite as described bythe manufacturer (PerSeptive Biosystems P/N GEN080020). Themomomethoxytrityl (MMT) group was removed by performing two standarddeblocking cycles on the DNA synthesizer.

The MEMSYN® device was then transferred to a prototype PNA synthesizer.The remaining PNA coupling cycles were performed on the Prototypesynthesizer running a cycle optimized for delivery of reagents to thecolumn. Generally the coupling cycle was as follows:

Reaction Step Time No. Description Reagent(s) (min) 1 Wash DMF/DCM (1:1)2 Purge column with Gas 0.167 3 Wait 0.167 4 Deblock Terminal Amino 25%DCA^(&) in DCM 2 Protecting Group 5 Repeat Step 4:1 time 6 Purge columnwith Gas 0.167 7 Wash DMF/DCM (1:1) 8 Purge column with Gas 0.167 9Neutralize 10% Pyridine in DMF 10 Wash DMF/DCM (1:1) 11 Deliver MonomerActivated Synthon Couping Mixture ® 12 Couple Activated Synthon Couping8 Mixture (Stop Flow) 13 Repeat Steps 11-12:1 time 14 Wash DMF/DCM (1:1)15 Repeat Steps 1-14 until the polymer is assembled ^(&)DichloroaceticAcid (DCA) ® Activated Synthon Couping Mixture comprised PNA synthon,HATU, and N-methylmorpholine in a molar ratio of approximately(0.1M:0.08M:0.2M)

The final DMBhoc group was not removed after synthesis. IT is essentialthat the terminal amino group remain protected until after the ammoniumhydroxide deprotection of the side chain protecting groups. Prematureremoval will result in partial degradation of the PNA portion of theoligomer.

Cleavage:

The MEMSYN® device was disassembled and the two membrane synthesissupports were removed. One of the membranes was cut into quarters. Toone quarter was added 2-3 drops of TFA. The strong yellow colorindicated the presence of the cation of DMBhoc. This membrane piece wasthen washed with DCM and treated with ninhydrin. The purple colorindicated the presence of terminal amino groups as expected.

The remaining synthesis membrane was added to a sealable centrifugetube. To the tube was added 1 mL of concentrated ammonium hydroxide. Thetube was tightly sealed and heated to 55° C. for 6 hr. The ammoniumhydroxide solution was removed and concentrated to dryness on a speedvac evaporator. The residue was redissolved in 200 μL of 50 millimolarammonium acetate buffer pH 7.

Reverse phase HPLC anaylsis of the crude oligomer is represented in FIG.11. The major product observed was purified by collecting the peak as iteluted from the HPLC detector. Purity of the isolated fraction wasconfirmed by reanalysis of the isolated product by HPLC. Thechromatogram of the product is depicted in FIG. 12. The impurity peaksobserved in the reanalysis chromatogram were confirmed to be bufferimpurities by running the gradient without injection of sample (see FIG.13). Further, the isolated product was analyzed by Matrix Assisted LaserDesoption Ionization-time of flight (MALDI-TOF) Mass Spectrometry on aprototype mass spectrometer. Calculated molecular weight was 3184.7 amu,mass found was (M-H) 3183.7 amu (within the error of the massspectrometric apparatus).

3 7 amino acids amino acid single linear not provided Peptide 1..7/note= “WHEREIN EACH XAA OF THE PNA SEQUENCE IS AMINOETHYLGLYCINEMODIFIED AS DESCRIBED IN THE SPECIFICATION” 1 Xaa Xaa Xaa Xaa Xaa XaaXaa 1 5 10 amino acids amino acid single linear not provided Peptide1..10 /note= “WHEREIN EACH XAA OF THE PNA SEQUENCE IS AMINOETHYLGLYCINEMODIFIED AS DESCRIBED IN THE SPECIFICATION” 2 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 1 5 10 5 amino acids amino acid single linear notprovided Peptide 1..5 /note= “WHEREIN EACH XAA OF THE PNA SEQUENCE ISAMINOETHYLGLYCINE MODIFIED AS DESCRIBED IN THE SPECIFICATION” 3 Xaa XaaXaa Xaa Xaa 1 5

What is claimed is:
 1. A PNA synthon having the formula:

wherein each of A₁-A₁₀ is the same or different and is independentlyselected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, hydroxy, methoxy, ethoxy, amide and ester groups; R⁴ isselected from hydrogen, methyl, and ethyl; R7 is selected from hydrogenand a side chain of a protected or unprotected naturally occurring (xamino acid; each of j, g, and h is the same or different and isindependently zero or an integer from one to five; and Ba is anucleobase wherein if said nucleobase has an exocyclic amino group, thensaid exocyclic amino group is protected by a base labile aminoprotecting group having the formula:

wherein W is selected from —CN and —S(O)_(n)R where n is one or two andR is (C₁-C₆) alkyl or (C₆-C₂₀) aryl; and each of R²-R⁴ is the same ordifferent and is independently selected from hydrogen, methyl, ethyl,n-propyl, isopropyl, n-butyl and t-butyl.
 2. The PNA synthon of claim 1wherein Ba is selected from thymidine, uridine, cytosine,pseudoisocytosine, 5-methyl-cytosine, isocytosine, adenine,7-deazaguanine, 7-deaza-8-azaguanine, and 2,6-diaminopurine.
 3. The PNAsynthon of claim 1 wherein g and j are one; h is zero; and R⁷ ishydrogen.
 4. The PNA synthon of claim 1 wherein R⁴ is hydrogen.
 5. ThePNA synthon of claim 1 wherein A₁, A₂, A₄, A₅, A₆, A₇, A₉, and A₁₀ ishydrogen; and A₃ and A₈ are methyl.
 6. The PNA synthon of claim 1wherein the base labile protecting group has the formula:


7. The PNA synthon of claim 1 wherein the base labile protecting grouphas the formula:


8. The PNA synthon of claim 1 wherein the base labile protecting grouphas the formula:


9. The PNA synthon of claim 1 having the formula:


10. The PNA synthon of claim 9 having the formula:


11. The PNA synthon of claim 10 wherein Ba has the formula:


12. The PNA synthon of claim 10 wherein Ba has the formula:


13. The PNA synthon of claim 10 wherein Ba has the formula:


14. The PNA synthon of claim 10 wherein Ba has the formula:


15. The PNA synthon of claim 10 wherein Ba has the formula:


16. The PNA synthon of claim 10 wherein Ba has the formula:


17. The PNA synthon of claim 10 wherein Ba has the formula: